Site-specific isotopic labeling of 1,4-diene systems

ABSTRACT

Methods for preparing isotopically modified 1,4-diene systems from non-isotopically modified 1,4-dienes involve selective oxidation of one or more bis-allylic position(s), or the preparation of isotopically modified 1,4-diene systems via trapping pi-allylic complexes with a source of deuterium or tritium. Such methods are useful for preparing isotopically modified polyunsaturated lipid including polyunsaturated fatty acids and polyunsaturated fatty acid derivatives.

INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

Any and all priority claims identified in the Application Data Sheet, orany correction thereto, are hereby incorporated by reference. Thisapplication is a continuation of U.S. application Ser. No. 15/778,182,filed May 22, 2018, which is the national phase under 35 U.S.C. § 371 ofprior PCT International Application No. PCT/US2016/051119 which has anInternational filing date of Sep. 9, 2016, which designates the UnitedStates of America, and which claims the benefit of U.S. ProvisionalApplication No. 62/258,993, filed Nov. 23, 2015. The aforementionedapplication is incorporated by reference herein in its entirety, and ishereby expressly made a part of this specification.

BACKGROUND Field

Isotopically modified polyunsaturated lipids, mixture of isotopicallymodified polyunsaturated lipids, methods of making such compounds ormixture, pharmaceutical compositions and medicaments comprising suchcompounds or mixtures, and method of using such compounds or mixtures totreat, prevent, alleviate, or diagnose disease, disorders, or conditionsare provided. Isotopically modified 1,4-diene systems such aspolyunsaturated fatty acids (“PUFAs”) are also disclosed.

Description of the Related Art

Oxidative damage is implicated in a wide variety of diseases including,but not limited to, mitochondrial diseases, neurodegenerative diseases,neurodegenerative muscle diseases, retinal diseases, energy processingdisorders, kidney diseases, hepatic diseases, lipidemias, cardiacdiseases, inflammation, and genetic disorders.

While the number of diseases associated with oxidative stress arenumerous and diverse, it is well established that oxidative stress iscaused by disturbances to the normal redox state within cells. Animbalance between routine production and detoxification of reactiveoxygen species (“ROS”) such as peroxides and free radicals can result inoxidative damage to cellular structures and machinery. Under normalconditions, a potentially important source of ROSs in aerobic organismsis the leakage of activated oxygen from mitochondria during normaloxidative respiration. Additionally, it is known that macrophages andenzymatic reactions also contribute to the generation of ROSs withincells. Because cells and their internal organelles are lipidmembrane-enveloped, ROSs can readily contact membrane constituents andcause lipid oxidation. Ultimately, such oxidative damage can be relayedto other biomolecules within the membrane and the cell, such as proteinsand DNA, through direct and indirect contact with activated oxygen,oxidized membrane constituents, or other oxidized cellular components.Thus, one can readily envision how oxidative damage may propagatethroughout a cell give the mobility of internal constituents and theinterconnectedness of cellular pathways.

Lipid-forming fatty acids are well-known as one of the major componentsof living cells. As such, they participate in numerous metabolicpathways, and play an important role in a variety of pathologies.Polyunsaturated Fatty Acids (“PUFAs”) are an important sub-class offatty acids. An essential nutrient is a food component that directly, orvia conversion, serves an essential biological function and which is notproduced endogenously or in large enough amounts to cover therequirements. For homeothermic animals, the two rigorously essentialPUFAs are linoleic (cis,cis-9,12-Octadecadienoic acid;(9Z,12Z)-9,12-Octadecadienoic acid; “LA”; 18:2;n-6) and alpha-linolenic(cis,cis,cis-9,12,15-Octadecatrienoic acid;(9Z,12Z,15Z)-9,12,15-Octadecatrienoic acid; “ALA”; 18:3;n-3) acids,formerly known as vitamin F (Cunnane S C. Progress in Lipid Research2003; 42:544-568). LA, by further enzymatic desaturation and elongation,is converted into higher n-6 PUFAs such as arachidonic (AA; 20:4;n-6)acid; whereas ALA gives rise to a higher n-3 series, including, but notlimited to, eicosapentaenoic acid (EPA; 20:5;n-3) and docosahexaenoic(DHA; 22:6;n-3) acid (Goyens P L. et al. Am. J. Clin. Nutr. 2006;84:44-53). Because of the essential nature of certain PUFAs or PUFAprecursors, there are many known instances of their deficiency and theseare often linked to medical conditions. Furthermore, many PUFAsupplements are available over-the-counter, with proven efficiencyagainst certain ailments (See, for example, U.S. Pat. Nos. 7,271,315 and7,381,558).

PUFAs endow mitochondrial membranes with appropriate fluidity necessaryfor optimal oxidative phosphorylation performance. PUFAs also play animportant role in initiation and propagation of the oxidative stress.PUFAs react with ROS through a chain reaction that amplifies an originalevent (Sun M, Salomon R G, J. Am. Chem. Soc. 2004; 126:5699-5708).However, non-enzymatic formation of high levels of lipid hydroperoxidesis known to result in several detrimental changes. Indeed, Coenzyme Q10has been linked to increased PUFA toxicity via PUFA peroxidation and thetoxicity of the resulting products (Do T Q et al, PNAS USA 1996;93:7534-7539). Such oxidized products negatively affect the fluidity andpermeability of their membranes; they lead to oxidation of membraneproteins; and they can be converted into a large number of highlyreactive carbonyl compounds. The latter include reactive species such asacrolein, malonic dialdehyde, glyoxal, methylglyoxal, etc.(Negre-Salvayre A, et al. Brit. J. Pharmacol. 2008; 153:6-20).

A logical way to obviate the damage associated with ROS would be toneutralize them with antioxidants. However, the success of antioxidanttherapies has so far been limited. This may be due to several reasons,including (1) the near-saturating amount of antioxidants already presentin living cells and the stochastic nature of the ROS inflicted damage,(2) the importance of ROS in cell signaling and hormetic (adaptive)upregulation of protective mechanisms, (3) the pro-oxidant nature ofsome antioxidants such as vitamin E, (4) the non-radical nature of PUFAperoxidation products, which can no longer be quenched with mostantioxidants.

SUMMARY

Some embodiments provide for a method of preparing isotopically modified1,4-diene systems comprising oxidizing a 1,4-diene at the bis-allylicposition to afford a peroxide; and inserting an isotope at the oxidizedbis-allylic position. In some embodiments, oxidizing a bis-allylicposition of a 1,4-diene utilizes a transition metal selected fromRhodium, Iridium, Nickel, Platinum, Palladium, Aluminum, Titanium,Zirconium, Hafnium, or Ruthenium. In other embodiments, the transitionmetal is a rhodium(II) metal or a ruthenium(III) metal. In someembodiments, inserting an isotope at the oxidized bis-allylic positionfurther comprises reducing a peroxide at the bis-allylic position toafford an alcohol. In other embodiments, amalgamated aluminum or aphosphine reduces a peroxide. In some embodiments, inserting an isotopeat an oxidized bis-allylic position further comprises exchanging analcohol with an isotope. In other embodiments, tributyltin deuterideexchanges an alcohol with deuterium.

Some embodiments provide for a method of preparing isotopically modified1,4-diene systems comprising oxidizing an alcohol at a bis-allylicposition to afford a ketone; and reducing the ketone to afford anisotopically substituted methylene group at the bis-allylic position. Inother embodiments, reducing a ketone utilizes Wolff-Kishner reactionconditions.

Some embodiments provide for a method of preparing isotopically modified1,4-diene systems comprising forming one or more pi-allylic complexesbetween a 1,4-diene and a metal; and inserting one or more isotopes inone or more bis-allylic positions. In some embodiments, the metal isselected from Ni, Pd and Ir. In other embodiments, the one or morepi-allylic complexes are formed as six-membered rings. In someembodiments, two or more pi-allylic complexes are formed as six-memberedrings. In other embodiments, the isotope is one or more deuterium atoms.

In some embodiments, any one or more of the chemical transformations canbe repeated to introduce one or more isotopes at one or more bis-allylicpositions.

In some embodiments, the 1,4-diene system is a PUFA. In otherembodiments, the PUFA is a compound of Formula 1A, 1B, or 1C, wherein R⁵is a C_(a)-C_(b) alkyl group wherein “a” and “b” of the C_(a)-C_(b) isany one or more of 1, 2, 3, 4, or 5.

In some embodiments, the PUFA is a compound of Formula 1A and R⁵ is aC₁-C₄ alkyl group.

Some embodiments relate to a method for site-specifically modifying apolyunsaturated lipid with an isotope, the method comprising reacting apolyunsaturated lipid with an isotope-containing agent in a presence ofa transition metal-based catalyst, whereby an isotopically-modifiedpolyunsaturated lipid having the isotope at one or more mono-allylic orbis-allylic sites is obtained, wherein the isotope-containing agentcomprises at least one isotope selected from the group consisting ofdeuterium, tritium, and combinations thereof.

Some embodiments relate to a method for site-specifically modifying apolyunsaturated lipid mixture with an isotope, the method comprisingreacting the polyunsaturated lipid mixture with an isotope-containingagent in a presence of a transition metal-based catalyst, whereby anisotopically-modified polyunsaturated lipid mixture having the isotopeat one or more mono-allylic or bis-allylic sites is obtained, whereinthe isotope-containing agent comprises at least one isotope selectedfrom the group consisting of deuterium, tritium, and combinationsthereof.

Some embodiments relate to a composition comprising one or moreisotopically-modified polyunsaturated lipids having an isotopepredominantly at one or more allylic sites, wherein the isotope isselected from the group consisting of deuterium, tritium, andcombinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a representation of a direct exchange method for isotopicallymodifying 1,4-diene systems

FIG. 2. is a schematic representation of methods to prepare isotopicallymodified 1,4-diene systems.

FIG. 3. is a schematic representation of the use of pi-allylic complexesand concomitant insertion of one or more isotopes to prepareisotopically modified 1,4-diene systems.

FIG. 4 shows a list of ruthenium based complexes tested for deuterationof the polyunsaturated lipid.

FIG. 5 shows an intermediate in the deuteration reaction at abis-allylic position of ethyl linolenate (E-lnn).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

As used herein, abbreviations are defined as follows:

-   ALA Alpha-linolenic acid-   LIN Linoleate-   LNN Linolenate-   ARA Arachidonate-   cap caprolactamate-   D⁻ Negatively charged deuterium ion-   T⁻ Negatively charged tritium ion-   DHA Docosahexaenoic acid-   DNA Deoxyribonucleic acid-   EPA Eicosapentaenoic acid-   HPLC High performance liquid chromatography-   IR Infrared-   LA Linoleic acid-   LC/MS Liquid Chromatography/Mass Spectrometry-   mg milligram-   mmol millimole-   NMR Nuclear magnetic resonance-   PUFAs Polyunsaturated fatty acids-   R_(f) Retention factor-   ROS Reactive oxygen species-   TBHP tert-butylhydroperoxide-   TLC Thin layer chromatography-   UV Ultraviolet-   Cp Cyclopentadienyl

As used herein, any “R” group(s) such as, without limitation, R¹, R²,R³, R⁴, R⁵, and R′ represent substituents that can be attached to theindicated atom. An R group may be substituted or unsubstituted.

As used herein, “C_(a) to C_(b)” in which “a” and “b” are integers referto the number of carbon atoms in an alkyl, alkenyl or alkynyl group, orthe number of carbon atoms in the ring of a cycloalkyl, cycloalkenyl,cycloalkynyl, aryl, heteroaryl or heteroalicyclyl group. That is, thealkyl, alkenyl, alkynyl, ring of the cycloalkyl, ring of thecycloalkenyl, ring of the cycloalkynyl, ring of the aryl, ring of theheteroaryl or ring of the heteroalicyclyl can contain from “a” to “b”,inclusive, carbon atoms. Thus, for example, a “C₁ to C₄ alkyl” grouprefers to all alkyl groups having from 1 to 4 carbons, that is, CH₃—,CH₃CH₂—, CH₃CH₂CH₂—, (CH₃)₂CH—, CH₃CH₂CH₂CH₂—, CH₃CH₂CH(CH₃)— and(CH₃)₃C—. If no “a” and “b” are designated with regard to an alkyl,alkenyl, alkynyl, cycloalkyl cycloalkenyl, cycloalkynyl, aryl,heteroaryl or heteroalicyclyl group, the broadest range described inthese definitions is to be assumed.

As used herein, “alkyl” refers to a straight or branched hydrocarbonchain that comprises a fully saturated (no double or triple bonds)hydrocarbon group. The alkyl group may have 1 to 20 carbon atoms(whenever it appears herein, a numerical range such as “1 to 20” refersto each integer in the given range; e.g., “1 to 20 carbon atoms” meansthat the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3carbon atoms, etc., up to and including 20 carbon atoms, although thepresent definition also covers the occurrence of the term “alkyl” whereno numerical range is designated). The alkyl group may also be a mediumsize alkyl having 1 to 10 carbon atoms. The alkyl group could also be alower alkyl having 1 to 6 carbon atoms. The alkyl group of the compoundsmay be designated as “C₁-C₄ alkyl” or similar designations. By way ofexample only, “C₁-C₄ alkyl” indicates that there are one to four carbonatoms in the alkyl chain, i.e., the alkyl chain is selected from methyl,ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl.Typical alkyl groups include, but are in no way limited to, methyl,ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, andhexyls. The alkyl group may be substituted or unsubstituted.

As used herein, “alkenyl” refers to an alkyl group that contains in thestraight or branched hydrocarbon chain one or more double bonds. Thealkenyl group may have 2 to 20 carbon atoms, although the presentdefinition also covers the occurrence of the term “alkenyl” where nonumerical range is designated. The alkenyl group may also be a mediumsize alkenyl having 2 to 9 carbon atoms. The alkenyl group could also bea lower alkenyl having 2 to 4 carbon atoms. The alkenyl group of thecompounds may be designated as “C₂-4 alkenyl” or similar designations.By way of example only, “C₂-4 alkenyl” indicates that there are two tofour carbon atoms in the alkenyl chain, i.e., the alkenyl chain isselected from the group consisting of ethenyl, propen-1-yl, propen-2-yl,propen-3-yl, buten-1-yl, buten-2-yl, buten-3-yl, buten-4-yl,1-methyl-propen-1-yl, 2-methyl-propen-1-yl, 1-ethyl-ethen-1-yl,2-methyl-propen-3-yl, buta-1,3-dienyl, buta-1,2,-dienyl, andbuta-1,2-dien-4-yl. Typical alkenyl groups include, but are in no waylimited to, ethenyl, propenyl, butenyl, pentenyl, and hexenyl, and thelike. An alkenyl group may be unsubstituted or substituted.

As used herein, “alkynyl” refers to an alkyl group that contains in thestraight or branched hydrocarbon chain one or more triple bonds. Thealkynyl group may have 2 to 20 carbon atoms, although the presentdefinition also covers the occurrence of the term “alkynyl” where nonumerical range is designated. The alkynyl group may also be a mediumsize alkynyl having 2 to 9 carbon atoms. The alkynyl group could also bea lower alkynyl having 2 to 4 carbon atoms. The alkynyl group of thecompounds may be designated as “C₂-4 alkynyl” or similar designations.By way of example only, “C₂-4 alkynyl” indicates that there are two tofour carbon atoms in the alkynyl chain, i.e., the alkynyl chain isselected from the group consisting of ethynyl, propyn-1-yl, propyn-2-yl,butyn-1-yl, butyn-3-yl, butyn-4-yl, and 2-butynyl. Typical alkynylgroups include, but are in no way limited to, ethynyl, propynyl,butynyl, pentynyl, and hexynyl, and the like. An alkynyl group may beunsubstituted or substituted.

As used herein, “cycloalkyl” refers to a completely saturated (no doubleor triple bonds) mono- or multi-cyclic hydrocarbon ring system. Whencomposed of two or more rings, the rings may be joined together in afused fashion. Cycloalkyl groups can contain 3 to 10 atoms in thering(s) or 3 to 8 atoms in the ring(s). A cycloalkyl group may beunsubstituted or substituted. Typical cycloalkyl groups include, but arein no way limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, and cyclooctyl. A cycloalkyl group may be unsubstituted orsubstituted.

As used herein, “cycloalkenyl” refers to a mono- or multi-cyclichydrocarbon ring system that contains one or more double bonds in atleast one ring; although, if there is more than one, the double bondscannot form a fully delocalized pi-electron system throughout all therings (otherwise the group would be “aryl,” as defined herein). Whencomposed of two or more rings, the rings may be connected together in afused fashion. A cycloalkenyl group may be unsubstituted or substituted.

As used herein, “cycloalkynyl” refers to a mono- or multi-cyclichydrocarbon ring system that contains one or more triple bonds in atleast one ring. If there is more than one triple bond, the triple bondscannot form a fully delocalized pi-electron system throughout all therings. When composed of two or more rings, the rings may be joinedtogether in a fused fashion. A cycloalkynyl group may be unsubstitutedor substituted.

As used herein, “carbocyclyl” refers to all carbon ring systems. Suchsystems can be unsaturated, can include some unsaturation, or cancontain some aromatic portion, or be all aromatic. A carbocyclyl groupmay be unsubstituted or substituted.

As used herein, “aryl” refers to a carbocyclic (all carbon) monocyclicor multicyclic aromatic ring system (including, e.g., fused, bridged, orspiro ring systems where two carbocyclic rings share a chemical bond,e.g., one or more aryl rings with one or more aryl or non-aryl rings)that has a fully delocalized pi-electron system throughout at least oneof the rings. The number of carbon atoms in an aryl group can vary. Forexample, the aryl group can be a C₆-C₁₄ aryl group, a C₆-C₁₀ aryl group,or a C₆ aryl group. Examples of aryl groups include, but are not limitedto, benzene, naphthalene, and azulene. An aryl group may be substitutedor unsubstituted.

As used herein, “heterocyclyl” refers to ring systems including at leastone heteroatom (e.g., O, N, S). Such systems can be unsaturated, caninclude some unsaturation, or can contain some aromatic portion, or beall aromatic. A heterocyclyl group may be unsubstituted or substituted.

As used herein, “heteroaryl” refers to a monocyclic or multicyclicaromatic ring system (a ring system having a least one ring with a fullydelocalized pi-electron system) that contain(s) one or more heteroatoms,that is, an element other than carbon, including but not limited to,nitrogen, oxygen, and sulfur, and at least one aromatic ring. The numberof atoms in the ring(s) of a heteroaryl group can vary. For example, theheteroaryl group can contain 4 to 14 atoms in the ring(s), 5 to 10 atomsin the ring(s) or 5 to 6 atoms in the ring(s). Furthermore, the term“heteroaryl” includes fused ring systems where two rings, such as atleast one aryl ring and at least one heteroaryl ring, or at least twoheteroaryl rings, share at least one chemical bond. Examples ofheteroaryl rings include, but are not limited to, furan, furazan,thiophene, benzothiophene, phthalazine, pyrrole, oxazole, benzoxazole,1,2,3-oxadiazole, 1,2,4-oxadiazole, thiazole, 1,2,3-thiadiazole,1,2,4-thiadiazole, benzothiazole, imidazole, benzimidazole, indole,indazole, pyrazole, benzopyrazole, isoxazole, benzoisoxazole,isothiazole, triazole, benzotriazole, thiadiazole, tetrazole, pyridine,pyridazine, pyrimidine, pyrazine, purine, pteridine, quinoline,isoquinoline, quinazoline, quinoxaline, cinnoline, and triazine. Aheteroaryl group may be substituted or unsubstituted.

As used herein, “heteroalicyclic” or “heteroalicyclyl” refers to three-,four-, five-, six-, seven-, eight-, nine-, ten-, up to 18-memberedmonocyclic, bicyclic, and tricyclic ring system wherein carbon atomstogether with from 1 to 5 heteroatoms constitute said ring system. Aheterocycle may optionally contain one or more unsaturated bondssituated in such a way, however, that a fully delocalized pi-electronsystem does not occur throughout all the rings. The heteroatoms areindependently selected from oxygen, sulfur, and nitrogen. A heterocyclemay further contain one or more carbonyl or thiocarbonylfunctionalities, so as to make the definition include oxo-systems andthio-systems such as lactams, lactones, cyclic imides, cyclicthioimides, and cyclic carbamates. When composed of two or more rings,the rings may be joined together in a fused fashion. Additionally, anynitrogens in a heteroalicyclic may be quaternized. Heteroalicyclyl orheteroalicyclic groups may be unsubstituted or substituted. Examples ofsuch “heteroalicyclic” or “heteroalicyclyl” groups include but are notlimited to, 1,3-dioxin, 1,3-dioxane, 1,4-dioxane, 1,2-dioxolane,1,3-dioxolane, 1,4-dioxolane, 1,3-oxathiane, 1,4-oxathiin,1,3-oxathiolane, 1,3-dithiole, 1,3-dithiolane, 1,4-oxathiane,tetrahydro-1,4-thiazine, 2H-1,2-oxazine, maleimide, succinimide,barbituric acid, thiobarbituric acid, dioxopiperazine, hydantoin,dihydrouracil, trioxane, hexahydro-1,3,5-triazine, imidazoline,imidazolidine, isoxazoline, isoxazolidine, oxazoline, oxazolidine,oxazolidinone, thiazoline, thiazolidine, morpholine, oxirane, piperidineN-Oxide, piperidine, piperazine, pyrrolidine, pyrrolidone, pyrrolidione,4-piperidone, pyrazoline, pyrazolidine, 2-oxopyrrolidine,tetrahydropyran, 4H-pyran, tetrahydrothiopyran, thiamorpholine,thiamorpholine sulfoxide, thiamorpholine sulfone, and their benzo-fusedanalogs (e.g., benzimidazolidinone, tetrahydroquinoline,3,4-methylenedioxyphenyl).

As used herein, “aralkyl” and “aryl(alkyl)” refer to an aryl groupconnected, as a substituent, via a lower alkylene group. The loweralkylene and aryl group of an aralkyl may be substituted orunsubstituted. Examples include but are not limited to benzyl,2-phenylalkyl, 3-phenylalkyl, and naphthylalkyl.

As used herein, “heteroaralkyl” and “heteroaryl(alkyl)” refer to aheteroaryl group connected, as a substituent, via a lower alkylenegroup. The lower alkylene and heteroaryl group of heteroaralkyl may besubstituted or unsubstituted. Examples include but are not limited to2-thienylalkyl, 3-thienylalkyl, furylalkyl, thienylalkyl, pyrrolylalkyl,pyridylalkyl, isoxazolylalkyl, and imidazolylalkyl, and theirbenzo-fused analogs.

A “(heteroalicyclyl)alkyl” is a heterocyclic or a heteroalicyclylicgroup connected, as a substituent, via a lower alkylene group. The loweralkylene and heterocyclic or a heterocyclyl of a (heteroalicyclyl)alkylmay be substituted or unsubstituted. Examples include but are notlimited tetrahydro-2H-pyran-4-yl)methyl, (piperidin-4-yl)ethyl,(piperidin-4-yl)propyl, (tetrahydro-2H-thiopyran-4-yl)methyl, and(1,3-thiazinan-4-yl)methyl.

“Lower alkylene groups” are straight-chained —CH₂— tethering groups,forming bonds to connect molecular fragments via their terminal carbonatoms. Examples include but are not limited to methylene (—CH₂—),ethylene (—CH₂CH₂—), propylene (—CH₂CH₂CH₂—), and butylene(—CH₂CH₂CH₂CH₂—). A lower alkylene group can be substituted by replacingone or more hydrogen of the lower alkylene group with a substituent(s)listed under the definition of “substituted.”

The amine ligands described herein can be monodentate or multidentateand include monoamine, diamine, and triamine moieties. Monoamines canhave the formula of N(R_(b))₂, and exemplary monoamines include but arenot limited to dialkylmonoamines (such as di-ra-butylamine, or DBA) andtrialkylmonoamines (such as N,N-dimethylbutylamine, or DMBA). Suitabledialkylmonoamines include dimethylamine, di-ra-propylamine,di-ra-butylamine, di-sec-butyl amine, di-tert-butylamine, dipentylamine,dihexylamine, dioctylamine, didecylamine, dibenzylamine,methylethylamine, methylbutylamine, dicyclohexylamine,N-phenylethanolamine, N-(p-methyl) phenylethanolamine, N-(2,6-dimethyl)phenylethanolamine, N-(p-chloro)phenylethanolamine, N-ethylaniline,N-butyl aniline, N-methyl-2-methylaniline, N-methyl-2,6-dimethylaniline,diphenylamine, and the like, and combinations thereof. Suitabletrialkylmonoamines include trimethylamine, triethylamine,tripropylamine, tributylamine, butyldimethylamine, phenyldiethylamine,and the like, and combinations thereof. Diamines can have the formula(R^(b))₂N—R^(a)—N(R^(b))₂, and exemplary diamines can includealkylenediamines, such as N,N′-di-ieri-butylethylenediamine, or DBEDA.Triamine refers to an organic molecule having three amine moieties,including but not limited to diethylene triamine (DETA), guanidine HCl,tetramethyl guanidine, and the like. For both the monoamine and diamineformula, R^(a) is a substituted or unsubstituted divalent residue; andeach R^(b) is independently hydrogen, C₁-C₈ alkyl, or C₆₋₁₀ aryl. Insome examples, of the above formula, two or three aliphatic carbon atomsform the closest link between the two diamine nitrogen atoms. Specificalkylenediamine ligands include those in which R^(a) is dimethylene(—CH₂CH₂—) or trimethylene (—CH₂CH₂CH₂—). R^(b) can be independentlyhydrogen, methyl, propyl, isopropyl, butyl, or a C₄-C₈ alpha-tertiaryalkyl group. In some embodiments, the diamine can be ethylenediamine. Insome embodiments, the triamine can be diethylenetriamine.

The alkylenediamine ligands can be monodentate or multidentate andexamples include N,N,N′,N′ tetramethylethylene diamine (TMED),N,N′-di-tert-butylethylenediamine (DBEDA),N,N,N′,N′-tetramethyl-1,3-diaminopropane (TMPD),N-methyl-1,3-diaminopropane, N,N′-dimethyl-1,3-diaminopropane,N,N,N′-dimethyl-1,3-diaminopropane, N-ethyl-1,3-diaminopropane,N-methyl-1,4-diaminobutane, N,N′-trimethyl-1,4-diaminobutane,N,N,N′-trimethyl-1,4-diaminobutane,N,N,N′,N′-tetramethyl-1,4-diaminobutane,N,N,N′,N′-tetramethyl-1,5-diaminopentane, and combinations thereof. Insome embodiments, the amine ligand is selected from di-ra-butylamine(DBA), N,N-dimethylbutylamine (DMBA), N,N′-di-tert-butylethylenediamine(DBEDA), and combinations thereof.

The alkene ligands described herein be monodentate or multidentate andinclude a molecule that has at least one non-aromatic carbon-carbondouble bond and can include but are not limited to monoalkene anddialkene. Examples of the alkene ligand can include ethylene, propylene,butene, hexene, decene, butadiene, and the like.

The isonitrile ligands described herein refer to a molecule having atleast one —NC moiety and can be monodentate or multidentate and includebut are not limited to monoisonitrile and diisonitrile ligands. Examplesof monoisonitrile and diisonitrile include but are not limited to C₁₋₁₀alkyl-NC and CN—R—NC and R is a C₁₋₁₀ alkylene, t-butyl-NC, methyl-NC,PhP(O)(OCH₂CH(t-Bu)NC)₂, PhP(O)(OCH₂CH(Bn)NC)₂ PhP(O)(OCH₂CH(i-Pr)NC)₂,PhP(O)(OCHCH₃CH(i-Pr)NC)₂, PhP(O)(OCH₂CH(CH₃)NC)₂. Additional isonitrileligands can be found in Naik et al., Chem. Commun., 2010, 46, 4475-4477,which is incorporated herein by reference in its entirety.

The nitrile ligands described herein refer to a molecule having at leastone —CN moiety and can be monodentate or multidentate and include butare not limited to monoisonitrile and diisonitrile ligands. Examples ofmonoisonitrile and diisonitrile include but are not limited to C₁₋₁₀alkyl-CN and CN—R—CN and R is a C₁₋₁₀ alkylene, acetonitrile,1,3,5-cyclohexanetricarbonitrile, propionitrile, butyronitrile,glutaronitrile, pivalonitrile, capronitrile, (CH₂)₃CN, (CH₂)₄CN,(CH₂)₅CN. Additional nitrile ligands can be found in Lee et al.,Inorganic and Nuclear Chemistry letters, v10, 10 (October 1974) p.895-898, which is incorporated herein by reference in its entirety.

The ether ligands described herein refer to a molecule having at leastone R—O—R moiety wherein each R is independently an alkyl or arylradical and can be monodentate or multidentate and include monoether,diether, and triether ligands. Examples of the monoether, diether,triether, and other suitable ether include but are not limited todimethyl ether, diethyl ether, tetrahydrofuran, dioxane,dimethoxyethane, diethylene glycol dimethyl ether, polyethylene glycol,and anisole.

The thioether ligands described herein refer to a molecule having atleast one R—S—R moiety a wherein each R is independently an alkyl oraryl radical and can be monodentate or multidentate and includemonothioether, dithioether, and trithioether ligands. Examples of themonothioether, dithioether, and trithioether include but are not limitedto dimethylsulfide and methyl phenyl sulfide.

The imine ligands described herein refer to a molecule having at leastone carbon nitrogen double bond moiety and can be monodentate ormultidentate and include monoimine, diimine, and triimine ligands.Examples of imine ligand include but are not limited to1,2-ethanediimine, imidazolin-2-imine, 1,2-diketimine, dimethylglyoxime,o-phenylenediamine, 1,3-diketimines, and glyoxal-bis(mesitylimine).

The carbene ligands as described herein refers to compounds having atleast one divalent carbon atom with only six electrons in its valenceshell when not coordinated to a metal. This definition is not limited tometal-carbene complexes synthesized from carbenes, but is ratherintended to address the orbital structure and electron distributionassociated with the carbon atom that is bound to the metal. Thedefinition recognizes that the “carbene” may not technically be divalentwhen bound to the metal, but it would be divalent if it were detachedfrom the metal. Although many such compounds are synthesized by firstsynthesizing a carbene and then binding it to a metal, the definition isintended to encompass compounds synthesized by other methods that have asimilar orbital structure and electron configuration. Lowry &Richardson, Mechanism and Theory in Organic Chemistry 256 (Harper & Row,1976) defines “carbene” in a way that is consistent with the way theterm is used herein. The carbene ligands described herein can bemonocarbene, dicarbene, and tricarbene. Examples of carbene ligandsinclude but are not limited to1,10-dimethyl-3,30-methylenediimidazolin-2,20-diylidene,1,10-dimethyl-3,30-ethylenediimidazolin-2,20-diylidene,1,10-dimethyl-3,30-propylenediimidazolin-2,20-diylidene,1,10-dimethyl-3,30-methylenedibenzimidazolin-2,20-diylidene,1,10-dimethyl-3,30-ethylenedibenzimidazolin-2,20-diylidene,1,10-dimethyl-3,3 propylenedibenzimidazolin-2,20-diylidene,

and n is 1, 2, or 3, and

Additional carbene ligands can be found in Huynh et al., Journal ofOrganometallic Chemistry, v696, 21, (October 2011), p.3369-33′75, andMaity et al., Chem. Commun., 2013,49, 1011-101, which are incorporatedherein by reference in their entireties.

The pyridine ligands as described herein refer to a molecule having atleast one pyridine ring moiety and can include monopyridine, dipyridine,and tripyridine ligands. Examples of the pyridine ligand include but arenot limited to 2,2′-bypiridine, and 2,6-Di(2-pyridyl) pyridine.

The phosphine ligands as described herein refer to a molecule having atleast one P(R⁴)₃, and each R⁴ is independently selected from the groupconsisting of hydrogen, optionally substituted C₁₋₁₅ alkyl, optionallysubstituted C₃₋₈ cycloalkyl, optionally substituted C₆₋₁₅ aryl, andoptionally substituted 4-10 membered heteroaryl. The phosphine ligandcan include monophosphine, bisphosphine, and trisphosphine. Examples ofsuitable phosphine ligand can include but are not limited to PH₃,trimethylphosphine, triphenylphosphine, methyldiphenylphosphine,trifluorophosphine, trimethylphosphite, triphenylphosphite,tricyclohexylphosphine, dimethylphosphinomethane (dmpm),dimethylphosphinoethane (dmpe), PROPHOS, PAMP, DIPAMP, DIOP, DuPHOS,P(tBu)₂Ph, 1,2-Bis(diphenylphosphino)ethane (dppe),1,1′-Bis(diphenylphosphino)ferrocene (dppf),4-(tert-butyl)-2-(diisopropylphosphaneyl)-1H-imidazole, P(t-Bu)₂(C₆H₅).

As used herein, a substituted group is derived from the unsubstitutedparent group in which there has been an exchange of one or more hydrogenatoms for another atom or group. Unless otherwise indicated, when agroup is deemed to be “substituted,” it is meant that the group issubstituted with one or more substituents independently selected fromC₁-C₆ alkyl, C₁-C₆ alkenyl, C₁-C₆ alkynyl, C₁-C₆ heteroalkyl, C₃-C₇carbocyclyl (optionally substituted with halo, C₁-C₆ alkyl, C₁-C₆alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy),C₃-C₇-carbocyclyl-C₁-C₆-alkyl (optionally substituted with halo, C₁-C₆alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), 5-10membered heterocyclyl (optionally substituted with halo, C₁-C₆ alkyl,C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), 5-10 memberedheterocyclyl-C₁-C₆-alkyl (optionally substituted with halo, C₁-C₆ alkyl,C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), aryl (optionallysubstituted with halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, andC₁-C₆ haloalkoxy), aryl(C₁-C₆)alkyl (optionally substituted with halo,C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), 5-10membered heteroaryl (optionally substituted with halo, C₁-C₆ alkyl,C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), 5-10 memberedheteroaryl(C₁-C₆)alkyl (optionally substituted with halo, C₁-C₆ alkyl,C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), halo, cyano,hydroxy, C₁-C₆ alkoxy, C₁-C₆ alkoxy(C₁-C₆)alkyl (i.e., ether), aryloxy,sulfhydryl (mercapto), halo(C₁-C₆)alkyl (e.g., —CF₃), halo(C₁-C₆)alkoxy(e.g., —OCF₃), C₁-C₆ alkylthio, arylthio, amino, amino(C₁-C₆)alkyl,nitro, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido,N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, acyl,cyanato, isocyanato, thiocyanato, isothiocyanato, sulfinyl, sulfonyl,and oxo (═O). Wherever a group is described as “substituted” that groupcan be substituted with the above substituents.

In some embodiments, substituted group(s) is (are) substituted with oneor more substituent(s) individually and independently selected fromC₁-C₄ alkyl, amino, hydroxy, and halogen.

It is to be understood that certain radical naming conventions caninclude either a mono-radical or a di-radical, depending on the context.For example, where a substituent requires two points of attachment tothe rest of the molecule, it is understood that the substituent is adi-radical. For example, a substituent identified as alkyl that requirestwo points of attachment includes di-radicals such as —CH₂—, —CH₂CH₂—,—CH₂CH(CH₃)CH₂—, and the like. Other radical naming conventions clearlyindicate that the radical is a di-radical such as “alkylene” or“alkenylene.”

The term “polyunsaturated lipid,” as used herein, refers to a lipid thatcontains one or more unsaturated bonds, such as a double or a triplebond, in its hydrophobic tail. The polyunsaturated lipid here can be apolyunsaturated fatty acid, polyunsaturated fatty acid ester,polyunsaturated fatty acid thioester, polyunsaturated fatty acid amide,polyunsaturated fatty acid mimetic, or polyunsaturated fatty acidprodrug.

The term “mono-allylic site”, as used herein, refers to the position ofthe polyunsaturated lipid, such as polyunsaturated fatty acid or esterthereof, that corresponds to a methylene group attached to only onevinyl group and is not adjacent to two or more vinyl group. For example,the mono-allylic site in a (9Z,12Z)-9,12-Octadecadienoic acid (linoleicacid) include the methylene groups at carbon 8 and carbon 14 positions.

The term “bis-allylic site,” as used herein, refers to the position ofthe polyunsaturated lipid, such as polyunsaturated fatty acid or esterthereof, that corresponds to the methylene groups of 1,4-diene systems.Examples of polyunsaturated lipid having deuterium at one or morebis-allylic positions include but are not limited to11,11-dideutero-cis,cis-9,12-Octadecadienoic acid(11,11-dideutero-(9Z,12Z)-9,12-octadecadienoic acid; D2-LA); and11,11,14,14-tetradeutero-cis,cis,cis-9,12,15-octadecatrienoic acid(11,11,14,14-tetradeutero-(9Z,12Z,15Z)-9,12,15-octadecatrienoic acid;D4-ALA).

The term “pro-bis-allylic position,” as used herein, refers to themethylene group that becomes the bis-allylic position upon desaturation.Some sites which are not bis-allylic in the precursor PUFAs will becomebis-allylic upon biochemical transformation. The pro-bis-allylicpositions, in addition to deuteration, can be further reinforced bycarbon-13, each at levels of isotope abundance above thenaturally-occurring abundance level. For example, the pro-bis-allylicpositions, in addition to existing bis-allylic positions, can bereinforced by isotope substitution as shown below in Formula (2),wherein R¹ is alkyl, cation, or H; m=1-10; n=1-5; and p=1-10. In Formula(2), the position of the X atom represents the pro-bis-allylic position,while the position of the Y atom represents the bis-allylic position,and one or more of X¹, X², Y¹, or Y² atoms can be deuterium atoms.

Another example of a compound having bis-allylic and pro-bis-allylicpositions is shown in Formula (3), wherein any of the pairs of Y¹-Y^(n)and/or X¹-X^(m) represent the bis-allylic and pro-bis-allylic positionsof PUFAs respectively and these positions may contain deuterium atoms.

It is understood that, in any compound described herein having one ormore chiral centers, if an absolute stereochemistry is not expresslyindicated, then each center may independently be of R-configuration orS-configuration or a mixture thereof. Thus, the compounds providedherein may be enantiomerically pure, enantiomerically enriched, or maybe stereoisomeric mixtures, and include all diastereomeric, andenantiomeric forms. In addition it is understood that, in any compounddescribed herein having one or more double bond(s) generatinggeometrical isomers that can be defined as E or Z, each double bond mayindependently be E or Z a mixture thereof. Stereoisomers are obtained,if desired, by methods such as, stereoselective synthesis and/or theseparation of stereoisomers by chiral chromatographic columns.

Likewise, it is understood that, in any compound described, alltautomeric forms are also intended to be included.

As used herein, the term “thioester” refers to a structure in which acarboxylic acid and a thiol group are linked by an ester linkage orwhere a carbonyl carbon forms a covalent bond with a sulfur atom —COSR,wherein R may include hydrogen, C₁₋₃₀ alkyl (branched or straight) andoptionally substituted C₆₋₁₀ aryl, heteroaryl, cyclic, or heterocyclicstructure. “Polyunsaturated fatty acid thioester” refers to a structureP-COSR, wherein P is a polyunsaturated fatty acid described herein.

As used herein, the term “amide” refers to compounds or moieties thatcontain a nitrogen atom bound to the carbon of a carbonyl or athiocarbonyl group such as compounds containing —C(O)NR¹R² or —S(O)NNR¹R², and R¹ and R² can independently be C₁₋₃₀ alkyl (branched orstraight), optionally substituted C₆₋₁₀ aryl, heteroaryl, cyclic,heterocyclic, or C₁-20 hydroalkyl. “Polyunsaturated fatty acid amide”refers to a structure wherein the amide group is attached to thepolyunsaturated fatty acid described herein through the carbon of thecarbonyl moiety.

As used herein the term “prodrug” refers to a precursor compound thatwill undergo metabolic activation in vivo to produce the active drug. Itis well-known that carboxylic acids may be converted to esters andvarious other functional groups to enhance pharmacokinetics such asabsorption, distribution, metabolism, and excretion. Esters are awell-known pro-drug form of carboxylic acids formed by the condensationof an alcohol (or its chemical equivalent) with a carboxylic acid (orits chemical equivalent). In some embodiments, alcohols (or theirchemical equivalent) for incorporation into pro-drugs of PUFAs includepharmaceutically acceptable alcohols or chemicals that upon metabolismyield pharmaceutically acceptable alcohols. Such alcohols include, butare not limited to, propylene glycol, ethanol, isopropanol,2-(2-ethoxyethoxy)ethanol (Transcutol®, Gattefosse, Westwood, N.J.07675), benzyl alcohol, glycerol, polyethylene glycol 200, polyethyleneglycol 300, or polyethylene glycol 400; polyoxyethylene castor oilderivatives (for example, polyoxyethyleneglyceroltriricinoleate orpolyoxyl 35 castor oil (Cremophor®EL, BASF Corp.),polyoxyethyleneglycerol oxystearate (Cremophor®RH 40 (polyethyleneglycol40 hydrogenated castor oil) or Cremophor®RH 60 (polyethyleneglycol 60hydrogenated castor oil), BASF Corp.)); saturated polyglycolizedglycerides (for example, Gelucire® 35/10, Gelucire® 44/14, Gelucire®46/07, Gelucire® 50/13 or Gelucire® 53/10, available from Gattefosse,Westwood, N.J. 07675); polyoxyethylene alkyl ethers (for example,cetomacrogol 1000); polyoxyethylene stearates (for example, PEG-6stearate, PEG-8 stearate, polyoxyl 40 stearate NF, polyoxyethyl 50stearate NF, PEG-12 stearate, PEG-20 stearate, PEG-100 stearate, PEG-12distearate, PEG-32 distearate, or PEG-150 distearate); ethyl oleate,isopropyl palmitate, isopropyl myristate; dimethyl isosorbide;N-methylpyrrolidinone; paraffin; cholesterol; lecithin; suppositorybases; pharmaceutically acceptable waxes (for example, carnauba wax,yellow wax, white wax, microcrystalline wax, or emulsifying wax);pharmaceutically acceptable silicon fluids; sorbitan fatty acid esters(including sorbitan laurate, sorbitan oleate, sorbitan palmitate, orsorbitan stearate); pharmaceutically acceptable saturated fats orpharmaceutically acceptable saturated oils (for example, hydrogenatedcastor oil (glyceryl-tris-12-hydroxystearate), cetyl esters wax (amixture of primarily C14-C18 saturated esters of C14-C18 saturated fattyacids having a melting range of about 43°-47° C.), or glycerylmonostearate).

In some embodiments, the fatty acid pro-drug is represented by the esterP—B, wherein the radical P is a PUFA and the radical B is a biologicallyacceptable molecule. Thus, cleavage of the ester P—B affords a PUFA anda biologically acceptable molecule. Such cleavage may be induced byacids, bases, oxidizing agents, and/or reducing agents. Examples ofbiologically acceptable molecules include, but are not limited to,nutritional materials, peptides, amino acids, proteins, carbohydrates(including monosaccharides, disaccharides, polysaccharides,glycosaminoglycans, and oligosaccharides), nucleotides, nucleosides,lipids (including mono-, di- and tri-substituted glycerols,glycerophospholipids, sphingolipids, and steroids). In some embodiments,alcohols (or their chemical equivalent) for incorporation into pro-drugsof PUFAs include polyalcohols such as diols, triols, tetra-ols,penta-ols, etc. Examples of alcohol include methyl, ethyl, iso-propyl,and other alkyl alcohol. Examples of polyalcohols include ethyleneglycol, propylene glycol, 1,3-butylene glycol, polyethylene glycol,methylpropanediol, ethoxydiglycol, hexylene glycol, dipropylene glycolglycerol, and carbohydrates. Esters formed from polyalcohols and PUFAsmay be mono-esters, di-esters, tri-esters, etc. In some embodiments,multiply esterified polyalcohols are esterified with the same PUFAs. Inother embodiments, multiply esterified polyalcohols are esterified withdifferent PUFAs. In some embodiments, the different PUFAs are stabilizedin the same manner. In other embodiments, the different PUFAs arestabilized in different manners (such as deuterium substitution in onePUFA and ¹³C substitution in another PUFA). In some embodiments, the oneor more PUFAs is an omega-3 fatty acid and the one or more PUFAs is anomega-6 fatty acid. In some embodiments, the ester is an ethyl ester. Insome embodiments, the ester is a mono-, di- or triglyceride.

It is also contemplated that it may be useful to formulate PUFAs and/orPUFA mimetics and/or PUFA pro-drugs as salts for use in the embodiments.For example, the use of salt formation as a means of tailoring theproperties of pharmaceutical compounds is well known. See Stahl et al.,Handbook of pharmaceutical salts: Properties, selection and use (2002)Weinheim/Zurich: Wiley-VCH/VHCA; Gould, Salt selection for basic drugs,Int. J. Pharm. (1986), 33:201-217. Salt formation can be used toincrease or decrease solubility, to improve stability or toxicity, andto reduce hygroscopicity of a drug product.

Formulation of PUFAs and/or PUFA esters and/or PUFA mimetics and/or PUFApro-drugs as salts can include any PUFA salt described herein.

The term “polyunsaturated fatty acid mimetic,” as used herein, refers tocompounds that are structurally similar to naturally occurringpolyunsaturated fatty acid but are non-isotopically modified to preventhydrogen abstraction at the bis-allylic position. Various methods can beused to non-isotopically modify the polyunsaturated fatty acid toproduce the polyunsaturated fatty acid mimetic, and examples include butare not limited to moving unsaturated bonds to eliminate one or morebis-allylic positions, replacing at least one carbon atom at thebis-allylic position with an oxygen or sulfur, replacing at least onehydrogen atom at the bis-allylic position with an alkyl group, replacingthe hydrogen atoms at the bis-allylic position with a cycloalkyl group,and replacing at least one double bond with a cycloalkyl group.

In some embodiments, the non-isotopic modification is achieved by movingunsaturated bonds to eliminate one or more bis-allylic positions. Thepolyunsaturated fatty acid can have the structure of Formula (I):

wherein R is H or C₁₋₁₀ alkyl, R¹ is H or C₁₋₁₀ alkyl, n is 1 to 4, andm is 1 to 12. In some embodiments, R¹ can be —C₃H₇. Examples of thepolyunsaturated fatty acid mimetic include but are not limited to:

In some embodiments, the non-isotopic modification is achieved byreplacing at least one carbon atom at the bis-allylic position with anoxygen or sulfur. The polyunsaturated fatty acid can have the structureof Formula (II):

wherein R is H or C₁₋₁₀ alkyl, R¹ is H or C₁₋₁₀ alkyl, X is O or S, n is1 to 4, and m is 1 to 12. In some embodiments, R¹ can be —C₃H₇. Examplesof the polyunsaturated fatty acid mimetic include but are not limitedto:

In some embodiments, the non-isotopic modification is achieved byreplacing at least one hydrogen atom at the bis-allylic position with analkyl group. The polyunsaturated fatty acid can have the structure ofFormula (III)

wherein R is H or C₁₋₁₀ alkyl, R¹ is H or C₁₋₁₀ alkyl, X is O or S, n is1 to 4, and m is 1 to 12. In some embodiments, R¹ can be —C₃H₇. Examplesof the polyunsaturated fatty acid mimetic include but are not limitedto:

In some embodiments, the non-isotopic modification is achieved byreplacing the hydrogen atoms at the bis-allylic position with acycloalkyl group. The polyunsaturated fatty acid can have the structureof Formula (IV):

wherein R is H or C₁₋₁₀ alkyl, R¹ is H or C₁₋₁₀ alkyl, n is 1 to 5, andm is 1 to 12. In some embodiments, R¹ can be —C₃H₇. Examples of thepolyunsaturated fatty acid mimetic include but are not limited to:

In some embodiments, the non-isotopic modification is achieved byreplacing at least one double bond with a cycloalkyl group. Thepolyunsaturated fatty acid can have the structure of Formula (V), (VI),or (VII)

wherein R is H or C₁₋₁₀ alkyl, R¹ is H or C₁₋₁₀ alkyl, n is 1 to 5, andm is 1 to 12. In some embodiments, R¹ can be —C₃H₇. Examples of thepolyunsaturated fatty acid mimetic include but are not limited to:

As used herein, “predominantly” refers to about 40% or greater. In oneembodiment, predominantly refers to greater than about 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%. In one embodiment,predominantly refers to about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95% or 100%. In one embodiment, predominantly refers toabout 50%-98%, 55%-98%, 60%-98%, 70%-98%, 50%-95%, 55%-95%, 60%-95%, or70%-95%. For example, “having an isotope predominantly at thebis-allylic site” means the amount of isotopic modification at thebis-allylic site is more than about 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95% or 100%. In another embodiment, “having anisotope predominantly at one or more allylic site” means the amount ofisotopic modification at the allylic site is more than about 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

“Subject” as used herein, means a human or a non-human mammal, e.g., adog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, a non-humanprimate or a bird, e.g., a chicken, as well as any other vertebrate orinvertebrate.

The term “mammal” is used in its usual biological sense. Thus, itspecifically includes, but is not limited to, primates, includingsimians (chimpanzees, apes, monkeys) and humans, cattle, horses, sheep,goats, swine, rabbits, dogs, cats, rodents, rats, mice guinea pigs, orthe like.

An “effective amount” or a “therapeutically effective amount” as usedherein refers to an amount of a therapeutic agent that is effective torelieve, to some extent, or to reduce the likelihood of onset of, one ormore of the symptoms of a disease or condition, and includes curing adisease or condition. “Curing” means that the symptoms of a disease orcondition are eliminated; however, certain long-term or permanenteffects may exist even after a cure is obtained (such as extensivetissue damage).

“Treat,” “treatment,” or “treating,” as used herein refers toadministering a compound or pharmaceutical composition to a subject forprophylactic and/or therapeutic purposes. The term “prophylactictreatment” refers to treating a subject who does not yet exhibitsymptoms of a disease or condition, but who is susceptible to, orotherwise at risk of, a particular disease or condition, whereby thetreatment reduces the likelihood that the patient will develop thedisease or condition. The term “therapeutic treatment” refers toadministering treatment to a subject already suffering from a disease orcondition.

The process of deuteration (or H/D exchange), which involves hydrogen(¹H) substitution with its heavier isotope deuterium (²H or D), can beapplied in nuclear magnetic resonance (NMR) spectroscopy, massspectrometry, polymer science, etc. Additionally, selective deuterationcan be a tool in pharmaceutical industry regarding drug design,development and discovery because metabolic pathway(s) of a certainpharmaceutical could be dramatically affected by H/D exchange. This canthen be used to, for instance, reduce the administered dosage becausethe biological half-life of a drug could be extended. Furthermore,certain drugs and biological molecules are also confronted by degradingmetabolic pathways leading to ruinous side effects that could be avertedby a specific H/D exchange process. For example, skin rush andhepatotoxicity in humans caused by Nevirpine (Viramune®), used for thetreatment of HIV infection, can be lessened with selective deuterationof this drug. The harmful metabolic pathway(s) of polyunsaturated fattyacids (PUFAs), molecules found in membranes of every cells and a feworganelles (subcellular parts), are associated with numerousneurological diseases such as Parkinson's, Alzheimer's, Friedreich'sataxia, etc. The deleterious metabolic pathways in PUFAs are usuallyinduced by radical-based molecules (radicals contain free electrons),which are constantly produced during the normal cellular oxygenconsumption process. These very reactive radical species then attack andcleave specific C—H bonds in PUFAs causing irreparable damage to thesebiological molecules, which could be prevented by selective H/D or H/Texchange. A drug based on a selectively deuterated PUFA, which waspreviously shown to have no serious side effects, can be used fortreatment of Friedreich's ataxia. For many other potentialpharmaceuticals, selective deuteration of PUFAs at the targetbis-allylic (a CH₂ group found between two alkene fragments), positionshas been limited to extensive synthetic procedures (or full syntheses)that might not be financially and practically viable at the industrialscale. Therefore, development of a selective and catalytic H/D or H/Texchange process, preferentially performed by a transition metal-basedcomplex, would be enormously valuable for further exploration andcommercial viability of these biologically important molecules.

The transition metal based catalysts and other agents for use in themethod described herein can include catalysts and agents described in J.W. Faller, H. Felkin, Organometallics 1985, 4, 1487; J. W. Faller, C. J.Smart, Organometallics 1989, 8, 602; B. Rybtchinski, R. Cohen, Y.Ben-David, J. M. L. Martin, D. Milstein, J. Am. Chem. Soc. 2003, 125,11041; R. Corberan, M. Sanaf, E. Peris, J. Am. Chem. Soc. 2006, 128,3974; S. K. S. Tse, P. Xue, Z. Lin, G. Jia, Adv. Synth. Catal. 2010,352, 1512; A. Di Giuseppe, R. Castarlenas, J. J. Perez-Torrente, F. J.Lahoz, V. Polo, L. A. Oro, Angew. Chem. Int. Ed. 2011, 50, 3938; M.Hatano, T. Nishimura, H. Yorimitsu, Org. Lett. 2016, 18, 3674; S. H.Lee, S. I. Gorelsky, G. I. Nikonov, Organometallics 2013, 32, 6599; G.Erdogan and D. B. Grotjahn, J. Am. Chem. Soc. 2009, 131, 10354; G.Erdogan and D. B. Grotjahn, Top Catal. 2010, 53, 1055; M. Yung, M. B.Skaddan, R. G. Bergman, J. Am. Chem. Soc. 2004, 126, 13033; M. H. G.Prechtl, M. Hölscher, Y. Ben-David, N. Theyssen, R. Loschen, D.Milstein, W. Leitner, Angew. Chem. Int. Ed. 2007, 46, 2269; T. Kurita,K. Hattori, S. Seki, T. Mizumoto, F. Aoki, Y. Yamada, K. Ikawa, T.Maegawa, Y. Monguchi, H. Sajiki, Chem. Eur. J. 2008, 14, 664; Y. Feng,B. Jiang, P. A. Boyle, E. A. Ison, Organometallics 2010, 29, 2857; S. K.S. Tse, P. Xue, C. W. S. Lau, H. H. Y. Sung, I. D. Williams, G. Jia,Chem. Eur. J. 2011, 17, 13918; E. Khaskin, D. Milstein, ACS Catal. 2013,3, 448; each of which is incorporated by reference herein in itsentirety.

Additional suitable transitional metal catalysts can include thosecatalysts described in D. B. Grotjahn, C. R. Larsen, J. L. Gustafson, R.Nair, A. Sharma, J. Am. Chem. Soc. 2007, 129, 9592; J. Tao, F. Sun, T.Fang, J. Organomet. Chem. 2012, 698, 1; Atzrodt, V. Derdau, T. Fey, J.Zimmermann, Angew. Chem. Int. Ed. 2007, 46, 7744. b) T. Junk, W. J.Catallo, Chem. Soc. Rev. 1997, 26, 401; L. Neubert, D. Michalik, S.Bähn, S. Imm, H. Neumann, J. Atzrodt, V. Derdau, W. Holla, M. Beller, J.Am. Chem. Soc. 2012, 134, 12239; T. G. Grant, J. Med. Chem. 2014, 57,3595; R. P. Yu, D. Hesk, N. Rivera, I. Pelczer, P. J. Chirik, Nature2016, 529, 195; all of which are incorporated by reference herein intheir interties.

Linoleic acid, the omega-6 essential PUFA that gives rise to higherhomologs such as arachidonic acid, has been successfully prepared as an11,11-D2-derivative by a 6-step synthesis (U.S. patent application Ser.No. 12/916,347). Methods for the synthesis of isotopically modified1,4-dienes such as PUFAs are described herein.

Synthesis of Isotopically Modified 1,4-Dienes:

Preparation of isotopically modified 1,4-diene systems at thebis-allylic position from non-modified 1,4-diene systems via a “directexchange” synthetic route represents an efficient method for thepreparation of compounds with isotopic modification at the bis-allylicposition. However, abstracting the bis-allylic hydrogen with base,quenching the resulting radical with D₂O, and then repeating the processto replace the second bis-allylic hydrogen will inevitably lead to adouble bond shift due to an intrinsic propensity of 1,4-diene systems torearrange into conjugated 1,3-dienes upon hydrogen abstraction from thebis-allylic position. A ‘softer’ method, one that does not result indouble bond rearrangement, is therefore required.

Some transition metals are known to weaken C—H (carbon-hydrogen) bonds.For example, platinum complexes can insert a platinum atom into a C—Hbond. The resultant organometalic compound is then amenable tosubsequent derivization to afford an isotopically labeled compound.However, the use of platinum as a transition metal, such is with aShilov system (Chem. Rev. 1997, 97(8), 2879-2932) may not be directlyapplicable to certain compounds, such as PUFAs because (1) the Shilovsystem preferentially activates stronger C—H bonds over weaker C—Hbonds, and (2) the platinum complexes are reactive towards double bonds.

In some embodiments, a direct exchange method affords a 1,4-diene systemthat is isotopically modified with one or more deuterium atoms and/orone of more tritium atoms at a bis-allylic position. Such an embodimentis represented in FIG. 1, where R¹, R², R³, and R⁴ are any one or moreof C_(a)-C_(b) alkyl, C_(a)-C_(b) alkenyl, C_(a)-C_(b) alkynyl,C_(a)-C_(b) cycloalkyl, C_(a)-C_(b) cycloalkenyl, C_(a)-C_(b)cycloalkynyl, C_(a)-C_(b) carbocyclyl, C_(a)-C_(b) heterocyclyl,C_(a)-C_(b) heteroaryl, C_(a)-C_(b) heteroalicyclic, C_(a)-C_(b)aralkyl, C_(a)-C_(b) heteroaralkyl, C_(a)-C_(b) heteroalicyclyl(alkyl),or a C_(a)-C_(b) lower alkylene group, wherein “a” and “b” of theC_(a)-C_(b) is any one or more of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, and Y is either deuterium or tritium.Each of R¹, R², R³, and R⁴ can be independently substituted orunsubstituted.

In some embodiments, the bis-allylic position of a 1,4-diene system isisotopically modified by treatment with a transition metal and anisotope source. In other embodiments, the transition metal is any one ormore of Rhodium, Iridium, Nickel, Platinum, Palladium, Aluminum,Titanium, Zirconium, Hafnium, or Ruthenium. In other embodiments thetransition metal is a rhodium(II) metal or a ruthenium(III) metal. Inother embodiments, the transition metal is dirhodium (II) orruthenium(III) and a ligand is utilized. In other embodiments, thetransition metal and ligand is a dirhodium (II) caprolactamate complexor a ruthenium(III) chloride complex. In some embodiments, thetransition metal is used in catalytic amounts. In other embodiments, thetransition metal is used in stoichiometric amounts. In some embodiments,a co-catalyst is used. In some embodiments, the isotope source is asource of D⁻ or T⁻. In other embodiments, the isotope source istributyltin deuteride.

In some embodiments, the synthetic route in FIG. 2 from a compound ofFormula 1 to compounds of Formulas 2 and 3 and/or a compound of Formula2 to a compound of Formula 3 involves proceeding through intermediatessuch as compounds of Formulas 4-6, wherein R′ is independently selectedfrom C_(a)-C_(b) alkyl, C_(a)-C_(b) alkenyl, C_(a)-C_(b) alkynyl,C_(a)-C_(b) cycloalkyl, C_(a)-C_(b) cycloalkenyl, C_(a)-C_(b)cycloalkynyl, C_(a)-C_(b) carbocyclyl, C_(a)-C_(b) heterocyclyl,C_(a)-C_(b) heteroaryl, C_(a)-C_(b) heteroalicyclic, C_(a)-C_(b)aralkyl, C_(a)-C_(b) heteroaralkyl, C_(a)-C_(b) heteroalicyclyl(alkyl),or a C_(a)-C_(b) lower alkylene group, wherein “a” and “b” of theC_(a)-C_(b) is any one or more of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20. Such embodiments are schematicallyrepresented in FIG. 2 with R¹, R², R³, R⁴, and Y having been previouslydefined.

In reaction (a) of FIG. 2, an adaptation of a method termed “allylicoxidation” is employed to afford a compound of Formula 5 from a compoundof Formula 1 (See Catino A J et al, JACS 2004; 126:13622; Choi H. et alOrg. Lett. 2007; 9:5349; and U.S. Pat. No. 6,369,247, the disclosures ofwhich are hereby incorporated by reference in their entirety). Oxidationof a compound of Formula 1 in the presence of a transition metal and anorganic peroxide readily affords an organic peroxide of Formula 5. Insome embodiments, the transition metal is any one or more of Rhodium,Iridium, Nickel, Platinum, Palladium, Aluminum, Titanium, Zirconium,Hafnium, or Ruthenium. In other embodiments the transition metal is arhodium(II) metal or a ruthenium(III) metal. In other embodiments, thetransition metal is dirhodium (II) or ruthenium(III) and a ligand isutilized. In other embodiments, the transition metal and ligand is adirhodium (II) caprolactamate complex or a ruthenium(III) chloridecomplex. In some embodiments, the transition metal is used in catalyticamounts. In other embodiments, the transition metal is used instoichiometric amounts.

Many organic peroxides can be used in the embodiments described herein.In some embodiments, these organic peroxides include C_(a)-C_(b) alkylperoxides, C_(a)-C_(b) alkenyl peroxides, C_(a)-C_(b) alkynyl peroxides,C_(a)-C_(b) cycloalkyl peroxides, C_(a)-C_(b) cycloalkenyl peroxides,C_(a)-C_(b) cycloalkynyl peroxides, C_(a)-C_(b) carbocyclyl peroxides,C_(a)-C_(b) heterocyclyl peroxides, C_(a)-C_(b) heteroaryl peroxides,C_(a)-C_(b) heteroalicyclic peroxides, C_(a)-C_(b) aralkyl peroxides,C_(a)-C_(b) heteroaralkyl peroxides, C_(a)-C_(b) heteroalicyclyl(alkyl)peroxides, or a C_(a)-C_(b) lower alkylene group peroxides, wherein “a”and “b” of the C_(a)-C_(b) is any one or more of 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20. In other embodiments, theorganic peroxide is TBHP.

In FIG. 2, the compound of Formula 5 represents a versatile intermediatefor the incorporation of deuterium and/or tritium at the bis-allylicposition of the 1,4-diene system. The compound of Formula 5 can bereduced to afford a compound of Formula 6 with an alcohol at thebis-allylic position. Such reductions of organic peroxides can beeffected with a variety of conditions that include, but are not limitedto hydrogen and a catalyst; LiAlH₄, Na in alcohol; Zn in acetic acid;CuCl; phosphines such as triphenyl phosphine and tributyl phosphine;H₂NCSNH₂; NaBH₄; SmI₂; and aluminium amalgam (See, e.g., ComprehensiveOrganic Transformations, 2^(nd) Ed., pages 1073-75 and the referencescited therein all of which are incorporated herein by reference).

In FIG. 2, the compound of Formula 6 also represents a versatileintermediate for the incorporation of deuterium and/or tritium at thebis-allylic position of the 1,4-diene system. A compound of Formula 6can be reduced to afford a compound of Formula 2 using multiple methods(reaction c), including, but not limited to, tributyltin deuteridedeoxygenation (Watanabe Y et al, Tet. Let. 1986; 27:5385); LiBDEt₃ (J.Organomet. Chem. 1978; 156,1,171; ibid. 1976; 41:18,3064); Zn/NaI (Tet.Lett. 1976; 37:3325); DCC (Ber. 1974; 107:4,1353); thioacetal (Tet.Lett. 1991; 32:49,7187). Repeating the steps described above forreactions a, b, and c in FIG. 2 can be used to transform themono-isotopically modified compound of Formula 2 into thedi-isotopically modified compound of Formula 3.

Alternatively, the compound of Formula 6 can be or further oxidized toafford a compound of Formula 4 with a bis-allylic carbonyl group(reaction d). Such oxidations can be effected with a variety ofconditions (See, e.g., Comprehensive Organic Transformations, 2^(nd)Ed., pages 1234-1250 and the references cited therein all of which areincorporated herein by reference). The carbonyl group present in thecompound of Formula 4 can be further reduced to the deuteromethylenegroup, —CD₂-, using various reaction conditions, that include, but arenot limited to, the Wolff-Kishner reaction (See, e.g., Furrow M E etal., JACS 2004; 126:5436 which is incorporated herein by reference).

Transition metals can also be employed to directly address thebis-allylic site of a 1,4-diene system such as the system present incompounds of Formulas 1 and 2 above. Such a use of transition metals isrepresented by reaction f of FIG. 2. The use of such transition metalscan involve the formation of a pi-allylic complex and concomitantinsertion of an isotope such as deuterium and/or tritium withoutre-arrangement of the double bonds. Embodiments of this process arerepresented in FIG. 3.

In FIG. 3, transition metal complexes such as compounds of Formula 7,including deuterium atom(s)-containing transition metal complexes suchas compounds of Formulas 8 and 9, assist in folding this 1,4-dienefragment into a six-membered ring system. The bis-allylic methylene atthe top of the six-membered structure can then be deuterated by analogywith the well-known process of deuterium scrambling in benzene. In someembodiments, M is any one or more of Rhodium, Iridium, Nickel, Platinum,Palladium, Aluminum, Titanium, Zirconium, Hafnium, or Ruthenium. Inother embodiments M is a rhodium(II) metal or a ruthenium(III) metal. Inother embodiments, M is dirhodium (II) or ruthenium(III) and a ligand isutilized. In other embodiments, M is a dirhodium (II) caprolactamatecomplex or a ruthenium(III) chloride complex. In FIG. 3, R¹, R², R³, R⁴,and Y are as defined above.

Synthesis of Isotopically Modified PUFAs:

Preparation of isotopically modified PUFAs from non-modified PUFAs via a“direct exchange” synthetic route can be accomplished as described abovein FIGS. 1-3. In some embodiments, compounds of Formula 1 are selectedfrom any one or more of the following compounds:

In the compounds of Formulas 1A-1C, R⁵ is a C_(a)-C_(b) alkyl,C_(a)-C_(b) alkenyl, C_(a)-C_(b) alkynyl, C_(a)-C_(b) cycloalkyl,C_(a)-C_(b) cycloalkenyl, C_(a)-C_(b) cycloalkynyl, C_(a)-C_(b)carbocyclyl, C_(a)-C_(b) heterocyclyl, C_(a)-C_(b) heteroaryl,C_(a)-C_(b) heteroalicyclic, C_(a)-C_(b) aralkyl, C_(a)-C_(b)heteroaralkyl, C_(a)-C_(b) heteroalicyclyl(alkyl), or a C_(a)-C_(b)lower alkylene group, wherein “a” and “b” of the C_(a)-C_(b) is any oneor more of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20. In some embodiments, R⁵ is a C_(a)-C_(b) alkyl group wherein“a” and “b” of the C_(a)-C_(b) is any one or more of 1, 2, 3, 4, or 5.

In some embodiments, compounds of Formula 2 are selected from any one ormore of the following compounds:

In the compounds of Formulas 2A-2C, Y and R⁵ are as previously defined.

In some embodiments compounds of Formula 3 are selected from any one ormore of the following compounds:

In the compounds of Formulas 3A-3E, Y and R⁵ are as previously defined.

In some embodiments, compounds of Formula 4 are selected from any one ormore of the following compounds:

In the compounds of Formulas 4A-4C, R⁵ is as previously defined.

In some embodiments, compounds of Formula 5 are selected from any one ormore of the following compounds:

In the compounds of Formulas 5A-5C, R¹ and R⁵ are as previously defined.

In some embodiments, compounds of Formula 6 are selected from any one ormore of the following compounds:

In the compounds of Formulas 6A-6C, R⁵ is as previously defined.

In some embodiments, compounds of Formula 7 are selected from any one ormore of the following compounds:

In the compounds of Formulas 7A-7C, M and R⁵ are as previously defined.

In some embodiments, compounds of Formula 8 are selected from any one ormore of the following compounds:

In the compounds of Formulas 8A-8C, M, Y and R⁵ are as previouslydefined.

In some embodiments, compounds of Formula 9 are selected from any one ormore of the following compounds:

In the compounds of Formulas 9A-9C, M, Y and R⁵ are as previouslydefined.

Method of Site-Specific Isotopic Modification

D-PUFAs can be manufactured by total synthesis, whereby simple fragmentsare chemically assembled in a step by step fashion to yield the desiredderivatives. The simple D-PUFA, D2-linoleic acid (D2-Lin), can be madeusing this approach. However, with increasing number of double bonds,the synthesis becomes more complex and expensive, giving lower yieldsand higher levels of impurities. D-PUFAs with the double bond numberhigher than 2, such as linolenic (LNN), arachidonic (ARA),eicosapentaenoic (EPA) and decosahexaenoic (DHA) are increasinglydifficult to produce. A synthetic method that does not require apurification step is highly desirable. But for the number of doublebonds higher than 2, the D-PUFAs would require an expensive and timeconsuming chromatographic purification step. For D-PUFAs with the numberof double bonds exceeding 4, the purification based on silver nitrateimpregnated silica gel chromatography is increasingly inefficient,rendering the total synthesis manufacturing approach essentiallyinadequate. The methods described herein not only achieves a selectiveand efficient isotopic modification with fewer reaction steps but alsoavoids expensive and time-consuming purification steps.

Conventional deuteration of molecules containing one alkene using thetransition metal as a catalyst often have problems, including thatpredominantly vinylic positions (hydrogen atom connected to a doublybonded carbon atom) are selectively deuterated. Many alkenes containmovement-restricted double bonds. Limited examples of linear(movement-unrestricted) alkenes yielded positional isomers, andcis-to-trans isomerisation always accompanied the deuteration processand lack of any reports on H/D exchange involving polyunsaturatedalkenes. Without wishing to be bound by any theory, it is believed thatsome catalytic systems are not adequate for the target H/D exchange atthe bis-allylic positions of PUFAs because the double bonds of thesemolecules are not only in the cis configuration but they are alsoseparated by a methylene group (i.e. bis-allylic position; FIG. 1). Thisparticular alkene arrangement is less thermodynamically favoured than asystem that would contain all trans-bonds in a conjugated configuration.Without wishing to be bound by any theory, it is believed that if acatalytic system is to perform selective deuteration at the bis-allylicpositions through any of the already described mechanisms, thepolyalkenes “falling” into these thermodynamic sinks/traps need to beprevented; otherwise, the target H/D exchange would need to be conductedthrough a new isotopic modification mechanism. Selective and efficientdeuteration of various polyalkenes (including PUFAs) at the bis-allylicsites by a commercially available Ru-based complex using the leastexpensive deuterium source D₂O can be achieved through the methodsdescribed herein. The isotopic modification described herein can occurin the absence of the thermodynamic side products (trans-isomers andconjugated alkenes) with a completely different mechanism from the onesalready established for deuteration of various organic substrate.

The methods described herein can be employed to achieve selective andefficient isotopic modification (e.g., D or T) of variouspolyunsaturated lipids (including PUFAs) at the bis-allylic sites by atransitional metal based catalyst (e.g., Ru-based complex) using aneasily available isotopic modification agent (e.g., D₂O as the deuteriumsource). The isotopic modification, such as the H/D exchange, can occurin the absence of the thermodynamic side products (trans-isomers andconjugated alkenes).

In addition, the methods described herein can be employed to performselective isotopic modification of a mixture of polyunsaturated lipids(e.g., PUFA or PUFA esters) without having to separate thepolyunsaturated lipids prior to reaction.

As described herein, the transition metal based catalysts (e.g. Ru basedcatalysts) can deuterate or tritiate bis-allylic positions of thesystems with three or more double bonds (e.g., E-Lnn, E-Ara, E-DHA,etc.) and cause no cis-trans isomerization or alkene conjugation in thepolyunsaturated lipid. An intermediate for the deuteration of abis-allylic position of E-Lnn is shown in FIG. 5.

The methods described herein can be employed to obtain polyunsaturatedlipid selectively deuterated or tritiated at one or more allylicpositions. In some embodiments, the method described herein can yield amixture of polyunsaturated lipid deuterated or tritiated at one or moreallylic positions.

A catalytic H/D exchange at the bis-allylic sites, starting directlyfrom non-deuterated, “natural” PUFAs, is achieved using the methodsdescribed herein. The synthesis methods described herein can solve boththermodynamic and selectivity challenges. PUFA's double bonds are notonly in cis configuration but they are also separated by a methylenegroup (i.e. bis-allylic positions, or skipped diene) which is lessthermodynamically favoured than a system that would contain all transbonds in a conjugated configuration. In addition, distinguishing betweenmono- and bis-allylic positions might be difficult if the mechanism forthe target H/D exchange may require the formation of an allylintermediate.

The methods described herein result in site-specific deuteration ofpolyunsaturated lipid, wherein the deuteration occurs at mon-allylic andbis-allylic positions. For polyunsaturated lipid having three or moredouble bonds, the method described herein can result in deuterationoccurring predominantly at the bis-allylic positions.

Some embodiments relate to a method for site-specifically modifying apolyunsaturated lipid with an isotope, comprising: reacting apolyunsaturated lipid with an isotope-containing agent in a presence ofa transition metal-based catalyst, whereby an isotopically-modifiedpolyunsaturated lipid having the isotope at one or more mono-allylic orbis-allylic sites is obtained, wherein the isotope-containing agentcomprises at least one isotope selected from the group consisting ofdeuterium, tritium, and combinations thereof.

In some embodiments, the polyunsaturated lipid is selected from thegroup consisting of a fatty acid, fatty acid ester, fatty acidthioester, fatty acid amide, fatty acid mimetic, and fatty acid prodrug.In some embodiments, the polyunsaturated lipid is selected from thegroup consisting of a fatty acid, fatty acid ester, fatty acid thioesterand fatty acid amide. In some embodiments, the polyunsaturated lipid isa fatty acid or fatty acid ester.

Polyunsaturated lipid having multiple double bonds can be isotopicallymodified using the methods described herein. In some embodiments, thepolyunsaturated lipid has two or more carbon-carbon double bonds. Insome embodiments, the polyunsaturated lipid has three or morecarbon-carbon double bonds.

In some embodiments, the polyunsaturated fatty acid has a structureaccording to Formula (IA):

wherein:

R¹ is selected from the group consisting of H and C₁₋₁₀ alkyl;

R² is selected from the group consisting of —OH, —OR³, —SR³, phosphate,and —N(R³)₂;

each R³ is independently selected from the group consisting of C₁₋₁₀alkyl, C₂₋₁₀ alkene, C₂₋₁₀ alkyne, C₃₋₁₀ cycloalkyl, C₆₋₁₀ aryl, 4-10membered heteroaryl, and 3-10 membered heterocyclic ring, wherein eachR³ is substituted or unsubstituted;

n is an integer of from 1 to 10; and

p is an integer of from 1 to 10.

In some embodiments, the polyunsaturated lipid is selected from thegroup consisting of omega-3 fatty acid, omega-6 fatty acid, and omega-9fatty acid. In some embodiments, the polyunsaturated lipid is an omega-3fatty acid. In some embodiments, the polyunsaturated lipid is an omega-6fatty acid. In some embodiments, the polyunsaturated lipid is an omega-9fatty acid.

In some embodiments, the polyunsaturated lipid is selected from thegroup consisting of linoleic acid and linolenic acid. In someembodiments, the polyunsaturated lipid is a linoleic acid. In someembodiments, the polyunsaturated lipid is a linolenic acid.

In some embodiments, the polyunsaturated lipid is selected from thegroup consisting of gamma linolenic acid, dihomo gamma linolenic acid,arachidonic acid, and docosatetraenoic acid.

In some embodiments, the polyunsaturated fatty acid ester is selectedfrom the group consisting of a triglyceride, a diglyceride, and amonoglyceride.

In some embodiments, the fatty acid ester is an ethyl ester.

In some embodiments, the isotopically-modified polyunsaturated lipid isa deuterated polyunsaturated lipid having deuterium at one or morebis-allylic sites.

In some embodiments, the isotopically-modified polyunsaturated lipid isa deuterated polyunsaturated lipid having deuterium at all bis-allylicsites.

In some embodiments, the isotopically-modified polyunsaturated lipid isa deuterated polyunsaturated lipid having deuterium at one or moremono-allylic sites.

In some embodiments, the polyunsaturated lipid have at least one1,4-diene moiety. In some embodiments, the polyunsaturated lipid havetwo or more 1,4-diene moieties.

In some embodiments, the isotopically-modified polyunsaturated lipid isa deuterated polyunsaturated lipid having a deuteration degree of morethan 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or99% at bis-allylic sites. In some embodiments, the isotopically-modifiedpolyunsaturated lipid is a deuterated polyunsaturated lipid having adeuteration degree of more than 50% at bis-allylic sites. In someembodiments, the isotopically-modified polyunsaturated lipid is adeuterated polyunsaturated lipid having a deuteration degree of morethan 90% at bis-allylic sites. In some embodiments, theisotopically-modified polyunsaturated lipid is a deuteratedpolyunsaturated lipid having a deuteration degree of more than 95% atbis-allylic sites. In some embodiments, the isotopically-modifiedpolyunsaturated lipid is a deuterated polyunsaturated lipid having adeuteration degree in the range of about 50% to about 95% at bis-allylicsites. In some embodiments, the isotopically-modified polyunsaturatedlipid is a deuterated polyunsaturated lipid having a deuteration degreein the range of about 80% to about 95% at bis-allylic sites. In someembodiments, the isotopically-modified polyunsaturated lipid is adeuterated polyunsaturated lipid having a deuteration degree in therange of about 80% to about 99% at bis-allylic sites.

In some embodiments, the isotopically-modified polyunsaturated lipid isa deuterated polyunsaturated lipid having a deuteration degree of lowerthan 80%, 70%, 60%, 50%, 45%, 40%, 35%, 30%, 20%, or 10% at mono-allylicsites. In some embodiments, the isotopically-modified polyunsaturatedlipid is a deuterated polyunsaturated lipid having a deuteration degreeof lower than 60% at mono-allylic sites. In some embodiments, theisotopically-modified polyunsaturated lipid is a deuteratedpolyunsaturated lipid having a deuteration degree of lower than 50% atmono-allylic sites. In some embodiments, the isotopically-modifiedpolyunsaturated lipid is a deuterated polyunsaturated lipid having adeuteration degree of lower than 45% at mono-allylic sites. In someembodiments, the isotopically-modified polyunsaturated lipid is adeuterated polyunsaturated lipid having a deuteration degree of lowerthan 40% at mono-allylic sites. In some embodiments, theisotopically-modified polyunsaturated lipid is a deuteratedpolyunsaturated lipid having a deuteration degree of lower than 35% atmono-allylic sites. In some embodiments, the isotopically-modifiedpolyunsaturated lipid is a deuterated polyunsaturated lipid having adeuteration degree of lower than 30% at mono-allylic sites. In someembodiments, the isotopically-modified polyunsaturated lipid is adeuterated polyunsaturated lipid having a deuteration degree of in therange of about 50% to about 20% at mono-allylic sites. In someembodiments, the isotopically-modified polyunsaturated lipid is adeuterated polyunsaturated lipid having a deuteration degree of in therange of about 60% to about 20% at mono-allylic sites.

In some embodiments, the transition metal-based catalyst comprises atransition metal selected from the group consisting of Rhodium, Iridium,Nickel, Platinum, Palladium, Aluminum, Titanium, Zirconium, Hafnium,Ruthenium, and combinations thereof. In some embodiments, the transitionmetal-based catalyst is a ruthenium catalyst.

In some embodiments, the transition metal-based catalyst has a structureaccording to Formula (IIA):

[ML¹(L²)_(m)]Q_(n)  (IIA)

wherein:

M is selected from the group consisting of Rhodium, Iridium, Nickel,Platinum, Palladium, Aluminum, Titanium, Zirconium, Hafnium, andRuthenium;

L¹ is selected from the group consisting of C₃₋₁₀ cycloalkyl, C₆₋₁₀aryl, 4-10 membered heteroaryl, and 3-10 membered heterocyclic ring,wherein L¹ is substituted or unsubstituted;

each L² is independently selected from the group consisting of amine,imine, carbene, alkene, nitrile, isonitrile, acetonitrile, ether,thioether, phosphine, pyridine, unsubstituted C₃₋₁₀ cycloalkyl,substituted C₃₋₁₀ cycloalkyl, substituted C₆₋₁₀ aryl, substituted 4-10membered heteroaryl, unsubstituted C₆₋₁₀ aryl, unsubstituted 4-10membered heteroaryl, substituted 3-10 membered heterocyclic ring,unsubstituted 3-10 membered heterocyclic ring and any combinationsthereof;

m is an integer of from 1 to 3,

Q is an anion bearing a single charge, and

n is 0 or 1.

In some embodiments, M is Ruthenium.

In some embodiments, L¹ is a C₃₋₁₀ cycloalkyl and L¹ is substituted orunsubstituted. In some embodiments, L¹ is a 4-10 membered heteroaryl andL¹ is substituted or unsubstituted. In some embodiments, L¹ is anunsubstituted cyclopentadienyl. In some embodiments, L¹ is a substitutedcyclopentadienyl.

In some embodiments, each L² is independently selected from the groupconsisting of amine, nitrile, isonitrile, acetonitrile, ether,thioether, phosphine, imine, carbene, pyridine, substituted C₆₋₁₀ aryl,substituted 4-10 membered heteroaryl, unsubstituted C₆₋₁₀ aryl,unsubstituted 4-10 membered heteroaryl, substituted 3-10 memberedheterocyclic ring, and unsubstituted 3-10 membered heterocyclic ring. Insome embodiments, each L² is —NCCH₃. In some embodiments, each L² isindependently selected from the group consisting of —NCCH₃, P(R⁴)₃, andsubstituted 4-10 membered heteroaryl, and any combinations thereof. Insome embodiments, at least one L² is —P(R⁴)₃, wherein each R⁴ isindependently selected from the group consisting of hydrogen, C₁₋₁₅alkyl, C₃₋₈ cycloalkyl, 4-10 membered heteroaryl, C₆₋₁₅ aryl, eachoptionally substituted with C₁₋₁₅ alkyl, C₂₋₁₅ alkene, C₂₋₁₅ alkyne,halogen, OH, cyano, alkoxy, C₃₋₈ cycloalkyl, 4-10 membered heteroaryl,and C₆₋₁₅ aryl. In some embodiments, P(R⁴)₃ is P(t-Bu)₂(C₆H₅). In someembodiments, P(R⁴)₃ is4-(tert-butyl)-2-(diisopropylphosphaneyl)-1H-imidazole. In someembodiments, each L² is independently acetonitrile or optionallysubstituted cyclopentadienyl.

In some embodiments, m is 2. In some embodiments, m is 3. In someembodiments, m is 4.

In some embodiments, Q is (PF₆)⁻, Cl⁻, F⁻, I⁻, Br⁻, NO₃ ⁻, ClO₄ ⁻, orBF₄ ⁻. In some embodiments, Q is (PF₆)⁻.

For the transition metal-based catalysts Formula (IIA) described herein,each L² can be independently selected from a list of suitablemonodentate or multidentate ligands. In some embodiments, each L² canindependently comprise at least two moieties selected from the groupconsisting of amine, imine, carbene, alkene, nitrile, isonitrile,acetonitrile, ether, thioether, phosphine, pyridine, substituted C₆₋₁₀aryl, substituted 4-10 membered heteroaryl, unsubstituted C₆₋₁₀ aryl,unsubstituted 4-10 membered heteroaryl, substituted 3-10 memberedheterocyclic ring, and unsubstituted 3-10 membered heterocyclic ring. Insome embodiments, one L² can be an amine, one L² can be a carbene, andone L² can be an imine. In some embodiments, at least one L² can havetwo or three chelating atoms in the ligand. In some embodiments, one L²in Formula (IIA) can be a ligand having both imine and phosphinemoieties and two or more chelating atoms. In some embodiments, one L² inFormula (IIA) can be a ligand having nitrile, isonitrile, and phosphinemoieties and at least three chelating atoms.

In some embodiments, the ruthenium catalyst has a structure selectedfrom the group consisting of:

In some embodiments, the ruthenium catalyst has a structure of:

In some embodiments, the transition metal based catalyst has a structureof

In some embodiments, the ruthenium catalyst has a structure of

Some embodiments relate to a method for site-specifically modifying apolyunsaturated lipid mixture with an isotope, the method comprisingreacting the polyunsaturated lipid mixture with an isotope-containingagent in a presence of a transition metal-based catalyst, whereby anisotopically-modified polyunsaturated lipid mixture having the isotopeat one or more mono-allylic or bis-allylic sites is obtained, whereinthe isotope-containing agent comprises at least one isotope selectedfrom the group consisting of deuterium, tritium, and combinationsthereof.

Compositions

Some embodiments relate to a composition comprising one or moreisotopically-modified polyunsaturated lipids having an isotopepredominantly at one or more allylic sites, wherein the isotope isselected from the group consisting of deuterium, tritium, andcombinations thereof. In some embodiments, the isotope is deuterium. Insome embodiments, the isotope is tritium.

In some embodiments, the isotopically modified polyunsaturated lipid isprepared according to the method described herein.

In some embodiments, the isotopically-modified polyunsaturated lipids inthe composition described herein are deuterated predominantly atbis-allylic sites. In some embodiments, the isotopically-modifiedpolyunsaturated lipids in the composition described herein aredeuterated predominantly at mono-allylic sites. In some embodiments, thecomposition described herein contains polyunsaturated lipid having twoor more carbon-carbon double bonds. In some embodiments, the compositiondescribed herein contains polyunsaturated lipid having three or morecarbon-carbon double bonds.

Isotopically labeled compounds afforded by the disclosed reactionschemes should have minimal or non-existent effects on importantbiological processes. For example, the natural abundance of isotopespresent in biological substrates implies that low levels of isotopicallylabeled compounds should have negligible effects on biologicalprocesses. Additionally, hydrogen atoms are incorporated into biologicalsubstrates from water, and is it known that the consumption of lowlevels of D₂O, or heavy water, does not pose a health threat to humans.See, e.g., “Physiological effect of heavy water.” Elements and isotopes:formation, transformation, distribution. Dordrecht: Kluwer Acad. Publ.(2003) pp. 111-112 (indicating that a 70 kg person might drink 4.8liters of heavy water without serious consequences). Moreover, manyisotopically labeled compounds are approved by the U.S. Food & DrugAdministration for diagnostic and treatment purposes.

Regarding isotopically labels compounds afforded by the disclosedreaction schemes, in some embodiments, deuterium has a natural abundanceof roughly 0.0156% of all naturally occurring hydrogen in the oceans onearth. Thus, a 1,4-diene system such as a PUFA having greater that thenatural abundance of deuterium may have greater than this level orgreater than the natural abundance level of roughly 0.0156% of itshydrogen atoms reinforced with deuterium, such as 0.02%, but preferablyabout 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 65%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 100%, or a range bounded by any two ofthe aforementioned percentages, of deuterium with respect to one or morehydrogen atoms in each PUFA molecule.

In some embodiments, isotopic purity refers to the percentage ofmolecules of an isotopically modified 1,4-diene system such as PUFAs inthe composition relative to the total number of molecules. For example,the isotopic purity may be about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 65%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or arange bounded by any two of the aforementioned percentages. In someembodiments, isotopic purity may be from about 50%-99% of the totalnumber of molecules in the composition.

In some embodiments, an isotopically modified compound may contain onedeuterium atom, such as when one of the two hydrogens in a methylenegroup is replaced by deuterium, and thus may be referred to as a “DI”compound. Similarly, an isotopically modified compound may contain twodeuterium atoms, such as when the two hydrogens in a methylene group areboth replaced by deuterium, and thus may be referred to as a “D2”compound. Similarly, an isotopically modified compound may contain threedeuterium atoms and may be referred to as a “D3” compound. Similarly, anisotopically modified compound may contain four deuterium atoms and maybe referred to as a “D4” compound. In some embodiments, an isotopicallymodified compound may contain five deuterium atoms or six deuteriumatoms and may be referred to as a “D5” or “D6” compound, respectively.

The number of heavy atoms in a molecule, or the isotopic load, may vary.For example, a molecule with a relatively low isotopic load may containabout 1, 2, 3, 4, 5, or 6 deuterium atoms. A molecule with a moderateisotopic load may contain about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20 deuterium atoms. In a molecule with a very high load, eachhydrogen may be replaced with a deuterium. Thus, the isotopic loadrefers to the percentage of heavy atoms in each molecule. For example,the isotopic load may be about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 65%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or arange bounded by any two of the aforementioned percentages, of thenumber of the same type of atoms in comparison to a molecule with noheavy atoms of the same type (e.g. hydrogen would be the “same type” asdeuterium). Unintended side effects are expected to be reduced wherethere is high isotopic purity in a composition, especially a PUFAcomposition, but low isotopic load in a given molecule. For example, themetabolic pathways will likely be less affected by using a PUFAcomposition with high isotopic purity but low isotopic load.

One will readily appreciate that when one of the two hydrogens of amethylene group is replaced with a deuterium atom, the resultantcompound may possess a stereocenter. In some embodiments, it may bedesirable to use racemic compounds. In other embodiments, it may bedesirable to use enantiomerically pure compounds. In additionalembodiments, it may be desirable to use diastereomerically purecompounds. In some embodiments, it may be desirable to use mixtures ofcompounds having enantiomeric excesses and/or diastereomeric excesses ofabout 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 65%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 100%, or a range bounded by any two ofthe aforementioned percentages. In some embodiments, it may bepreferable to utilize stereochemically pure enantiomers and/ordiastereomers of embodiments—such as when enzymatic reactions orcontacts with chiral molecules are being targeted for attenuatingoxidative damage. However, in many circumstances, non-enzymaticprocesses and/or non-chiral molecules are being targeted for attenuatingoxidative damage. In such circumstances, embodiments may be utilizedwithout concern for their stereochemical purity. Moreover, in someembodiments, mixtures of enantiomers and diastereomers may be used evenwhen the compounds are targeting enzymatic reactions and/or chiralmolecules for attenuating oxidative damage.

In some aspects, isotopically modified compounds impart an amount ofheavy atoms in a particular tissue upon administration. Thus, in someaspects, the amount of heavy molecules will be a particular percentageof the same type of molecules in a tissue. For example, the percentageof heavy molecules may be about 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, or a range bounded by the selection of any two of theaforementioned percentages.

EXAMPLES Example 1. Oxidation of Methyl Linoleate

Methyl linoleate (400 mg, 1.36 mmol; 99% purity) was dissolved in 5 mLdry methylene chloride. Potassium carbonate (94 mg, 0.68 mmol) and asmall crystal (2 mg) of dirhodium (II) caprolactamate were added and themixture was allowed to stir to afford a pale purple suspension.Tert-butylhydroperoxide (0.94 mL, 6.8 mmol; 70% aq. solution 7.2 M) wasadded and the reaction mixture was allowed to stir. TLC (9:1heptane:ethyl acetate) taken at 45 min showed the absence of startingmaterial (R_(f)=0.51), one major close running spot (R_(f)=0.45), and anumber of slower spots. The reaction mixture was allowed to stir in thepresence of 15% aqueous sodium sulfite, washed with 12% brine andsaturated brine, then dried over sodium sulfate. Filtration and removalof volatiles afforded a yellow substance. Four runs on this scale werecombined and chromatographed on a silica gel column (bed=2.4 cm×25 cm).The column was packed with 99:1 heptane:ethyl acetate and eluted with agradient of 1% to 7% ethyl acetate. The major product of R_(f)=0.45 wasisolated as 300 mg of a colorless substance that was substantially pureby TLC and LC/MS. The NMR spectra of this material matched that reportedfor methyl 11-t-butylperoxylinoleate (Lipids 2000, 35, 947). UV & IRanalysis confirmed that the isolated product was not a conjugated diene.

Oxidation of methyl linoleate with TBHP and catalytic ruthenium (III)chloride in heptane/water under the conditions specified in U.S. Pat.No. 6,369,247, example 3, which is incorporated herein by reference,gave essentially the same results as the reaction sequence describedabove.

Example 2. Synthesis of Isotopically Modified Polyunsaturated Lipid

Various Ru-based complexes were used for selectively performing isotopicmodification at the bis-allylic sites. Some of the tested PUFAs have twodouble bonds and some have three or more double bonds. The reactions hadno observable trans isomerisation nor the formation of conjugateconfigurations.

FIG. 4 shows the six Ru based complexes used for the isotopicmodification reactions. In FIG. 4, Complex 1 can perform catalyticdeuteration at allylic positions of mono-alkenes. However, this complexcan also be an excellent alkene zipper catalyst capable of moving adouble bond across, for up to 30 positions.

Various Ru complexes (Complex 1-6 in FIG. 4) were tested in thesite-specific isotopic modification reaction. In each test, thepolyunsaturated lipid was combined with an acetone solution containingeach complex, and the reaction mixture immediately proceeded to form ofa conjugated system. The results are shown in Table 1 below, which isproceeded by a key including definitions of abbreviations used in thetable.

TABLE 1 Deuteration of polyunsaturated lipids with ruthenium complexesshown in Figure 4. Extent of deuteration (%) Complex Double bond TimeYield Mono- Bis- # (%)^([b]) Substrate conjugation (h) (%) allylicallylic  1^([c]) 1 (5%) E-Lin Yes^([d]) (82%) 170 n.d. N/A^([e])N/A^([e])  2^([f]) 2 (5%) E-Lin No 48 n.i. 60  0  3^([f]) 3 (5%) E-LinNo 24 n.i. 90  0  4 4 (1%) E-Lin No 0.25 n.i. 87  0  5 4 (1%) E-Lnn No1 >99 19 (23, 15)^([g]) 94  6 4 (1%) E-Ara No 24 >99 13 (15, 10)^([g])96  7 4 (2%) E-DHA No 18 >99 26 (47, 5)^([g]) 96  8 4 (1%) T-Lnn No7 >99 22 95  9 4 (5%) T-Ara No 3 n.i. 17 95 10^([h]) 4 (3%) E-Lnn E-AraE-DHA No 1 n.i. 17 96 11 4 (1%) O-Lnn No 18 n.i. 21 (26, 16)^([g]) 96 124 (1%) H-Lnn No 18 n.i. 36 (46, 26)^([g]) 98 13 4 (2%) O-DHA No 18 n.i.47 (57, 38)^([g]) 98 14 4 (2%) H-DHA No 18 n.i. 30 (36, 24)^([g]) 98 155 (1%) E-Lnn No 24 n.d.  0  0 16 6 (1%) E-Lnn No^([i]) 24 n.d. N/A^([e])N/A^([e]) 17^([j]) 4 (2%) POLY No 24 n.i. 90 N/A 18^([k]) 4 (1%) HEXD No2 n.i. 95  0

n.d. = not determined; n.i. = not isolated. ^([a])equiv. of D₂O withrespect to a bis-allylic position. ^([b])% of complex with respect tothe substrate. ^([c])73 equiv. of D₂O used. ^([d])regardless of thepresence or absence of D₂O. ^([e])conjugation or cis/trans isomerisationprevented the estimation on the % deuteration. ^([f])73 equiv. of D₂Oand 60° C. ^([g])Combination of ¹H and ¹³C NMR spectroscopy allowed forestimation of the deuteration percentage at the aliphatic mono-allylicposition on one end and ester/alcohol/reduced-aliphatic mono-allylicposition on the other for select substrates. ^([h])1:1:1 mol ratio ofthe substrates. ^([i])cis-trans isomerisation observed with noconjugation. ^([j])5 equiv. of D₂O. ^([k])20 equiv. of D₂O.

The deuteration of the bis-allylic position of E-Lnn with complex 4 wasachieved by adding E-Lnn to an excess of D₂O in the acetone solution at60° C., obtaining ethyl linolenate (E-Lnn) as 94% bis-allylic and 19% ofmono-allylic protons underwent H/D exchange (entry 5, Table 1),signifying selective isotopic modification.

Complex 4 was tested in the deuteration reaction and proved to be anefficient catalyst for the site-specific deuteration than the otherinvestigated complexes tested. It is possible that the phosphine ligandin 1, 2 and 3 (including the imidazolyl moiety in 1) were not involvedin the deuteration process. Nevertheless, the presence of cyclopentylring seems to be quite important as complex 5 (FIG. 4) showed lowactivity towards E-Lnn (entry 7, Table 1). However, complex 6 (FIG. 4),which is a permethylated analogue of 4, was shown to perform onlycis-trans isomerization without any hints at the target deuteration whenE-Lnn was used as the substrate.

Ethyl arachidonate (E-Ara, entry 6, Table 1), ethyl docosahexaenoate(E-DHA, entry 7, Table 1), the triglycerides of linolenic (T-Lnn; entry8, Table 1) and arachidonic (T-Ara; entry 9, Table 1) acids weresuccessfully and selectively deuterated at bis-allylic positions withcomplex 4. It was also possible to perform the selective H/D exchangeusing a mixture of E-Lnn, E-Ara and E-DHA (mass ratio of 1:1:1, entry10, Table 1), signifying a great potential to eliminate costlyseparations among various PUFAs. The alcohol (O-Lnn and O-DHA; entries11 and 13) and hydrocarbon (H-Lnn and H-DHA; entries 12 and 14)analogues of E-Lnn and E-DHA were also adequate substrates for thetarget deuteration. The average deuteration at the bis-allylic positionwas around 95% while the mono-allylic positions were deuterated at about25% or less for select substrates. A higher degree of deuteration (about98%) at the bis-allylic position was possible but with loss ofselectivity with respect to the monoallylic positions (see, for example,entries 12 and 13, Table 1). By using ¹³C NMR spectroscopy, it waspossible to estimate the relative percentage of deuteration at different(aliphatic vs ester/alcohol/reduced-aliphatic) mono-allylic positions.In all cases, the mono-allylic sites with a longer chain or presence ofester/alcohol groups were deuterated to a lesser extent presumably dueto a higher steric influence (e.g. entry 7, Table 1).

Controlled experiments were performed by using H₂O instead of D₂O inorder to examine whether any cis-trans isomerisation occurs for thereaction conditions used in Table 1. It has been reported that anallylic position of E-Lin in ¹³C NMR spectra was downfield shifted byabout 5 ppm for each double bond that had been isomerized from cis totrans. For example, the bis-allylic positions in E-Lnn have two adjacentdouble bonds and hence if any one of these double bonds is isomerized totrans the δc signal would be downfield shifted by about 5 ppm. If bothbonds are isomerized to trans, then the shift is about 10 ppm. Usingthis information, experiments described in Table 2 wherein D₂O wasreplaced with H₂O were repeated, confirming by ¹³C NMR spectroscopy thatthere was no formation of any trans-containing isomer for any of thePUFAs attempted.

Without wishing to be bound by any theory, it is believed that theexperimental data collected thus far indicated that the mechanism ofdeuteration using complex 4 was different from the one described forother organic substrates. Considering that complex 4 (i) deuteratesE-Lin only at the mono-allylic positions, (ii) deuterates bis-allylicpositions of the systems with three or more double bonds (E-Lnn, E-Ara,E-DHA) and (iii) causes no cis-trans isomerisation in these PUFAs, itwas then likely that the anionic allylic intermediate was not involvedin the overall mechanism for the observed H/D exchange. Without wishingto be bound by any theory, it is believed that a possible intermediatefor the deuteration of a bis-allylic position of E-Lnn is shown in FIG.5. Without wishing to be bound by any theory, it is believed that thesubstrate binds to the ruthenium center through two double bonds, whichwould bring the protons of one of the bis-allylic sites closer to themetal center, creating a Ru H contact (agositc interaction). This Ru Hcontact would then increase the acidity of the proton, allowing for thetarget H/D exchange without any cis-trans isomerization or the formationof a conjugate system. This intermediate would lead to the mono-allylicselectivity for E-Lin, as the sole bis-allylic position of thissubstrate would be facing away from the ruthenium center. It would alsoresult in the deuteration selectivity of the mono-allylic positionsbased on the steric demand of the pendant groups, which was the case forE-DHA (entry 9, Table 1).

The direct bis-allylic deuteration method described herein efficientlymodified various PUFAs using a number of Ru-based complexes (FIG. 4).Complex (2), compared to Complex 1 gave no double bond conjugation butthis complex could only perform the deuteration of the mono-allylicpositions of E-Lin. However, using E-Lnn resulted in deuteration of thebis-allylic positions as well. The phosphine ligand in complexes 1, 2and 3, apart from the reaction rates, had no influence on the targetdeuterations leading to complex 4 being the most viable option. Lastly,H/D exchange at the bis-allylic position of E-Lnn, E-Ara, E-DHA andT-Lnn, with a minimal deuteration at the mono-allylic positions, wereachieved with complex 4.

The H/D exchange using polybutadiene and cis-1,4-hexadiene was alsotested. Even though the solubility of cis-polybutadiene was not ideal inthe acetone/D₂O mixture, there was evidence to suggest that this polymercould also be deuterated at the mono-allylic positions (POLY; entry 17,Table 1). As this material contains two methylene groups between thealkene fragments it indicated that the described deuteration was notlimited to only skipped alkenes (e.g. PUFAs). Furthermore, successfulH/D exchange was also performed at the allyl-CH₃ group ofcis-1,4-hexadiene (HEXD; entry 18, Table 1) emphasizing that theexistence of chemically different alkene groups could be used for thedeuteration.

The role of the Cp ligand in the Ruthenium catalyst was also studied.Hexa(acetonitrile) complex 5 (FIG. 4) showed no deuteration abilityusing E-Lnn signifying the importance of the cyclic substituent (entry15, Table 1). However, if the permethylated analogue was used (i.e.complex 6; FIG. 4), only cis-to-trans isomerisation was observed (entry16, Table 1). The rates of the cis-trans isomerisation ofpolyunsaturated alkenes progressively increased with sequential additionof methyl fragments to the Cp ring possibly due to the loosening of oneof the Ru-alkene bonding interactions as the Cp ring is methylated.

Without wishing to be bound by any theory, it is believed that Complex 1formed a conjugated system when E-Lin was used as the substrate, and itmight be ineffective for catalyzing the target bis-allylic deuterations.Forming a more sterically demanding complex (2) resulted in the absenceof double bond conjugation and selective deuteration of the mono-allylicpositions of E-Lin. Without wishing to be bound by any theory, it isbelieved that the redundancy of the entire imidazolyl-phosphine ligandwas supported by the activity of complex 3, and more importantly complexphosphine-free complex 4. Complex 4 was then used to perform selectivedeuteration of various substrates including polybutadiene andcis-1,4-hexadiene. Without wishing to be bound by any theory, it isbelieved that the mechanism may involve the formation of a bis-alkeneintermediate, which is unlike any other mechanism described fordeuteration of various organic substrates.

In most cases (e.g., entries 1-4 and 9-18, Table 1) the reaction wasprepared and monitored using a J. Young NMR tube according to thefollowing procedure: A J. Young NMR tube was charged with 10 mg of asubstrate followed by D₂O (73 or 100 equiv. per bis-allylic position; or5 equiv. for polybutadiene per methylene group due to solubility issues)and acetone-d₆ (˜0.5 ml) after which first ¹H NMR spectrum was acquired.Inside a glove box a ruthenium complex was dissolved in acetone-d₆ (˜0.3ml) and transferred in the tube and heated if necessary. Reactionprogress was monitored by hourly ¹H NMR scans during first 12 hoursfollowed by daily scans.

For select runs (entries 5-8, Table 1) the reaction was performed using100 mg of substrates to emphasize that virtually quantitative yields ofthese reactions could be obtained: Inside a glove box two scintillationvials were charged with a substrate (E-Lnn, E-Ara, E-DHA or T-Lnn) andcomplex 4, respectively. Both were transferred into two separate Schlenkflasks using three 0.5 ml acetone portions each. D₂O was added to theflask containing PUFA followed by the amount of acetone necessary toform a homogenous solution. Then a solution of complex 4 in acetone wastransferred to the substrate/D₂O-containing solution reaction was leftstirring at room temperature. Upon completion of the reaction, excess of2 N HCl (not less than 5 times volume of reaction mixture) was added andthe mixture was allowed to stir vigorously for 15 minutes. The productwas extracted with 100 ml of hexane and the solution was then washedwith saturated NaHCO₃ and NaCl solutions and dried over anhydrous NaSO₄.The solution was filtered and activated carbon was added. Stirring foranother 15 minutes, filtration and removal of volatiles in vacuoafforded desired product.

General Procedure A for Deuteration of E-Lin and E-Lnn with VariousRuthenium Complexes (Table 1)

A J Young NMR tube was charged with PUFA followed by D₂O and acetone-d₆,after which first ¹H NMR spectrum was acquired. Inside a glove box, PUFAsolution was then transferred into a scintillation vial containingrespective ruthenium complex. The resulting solution was thoroughlymixed and transferred back into the NMR tube. Reaction progress wasmonitored by hourly ¹H NMR scans during first 12 hours followed by dailyscans.

General Procedure B for Deuteration of Various PUFAs Using Complex 4

Inside a glove box two scintillation vials were charged with PUFA andcomplex 4, respectively. Both were transferred into two separate Schlenkflasks using three 0.5 ml acetone portions each. D₂O was added to theflask containing PUFA followed by the amount of acetone necessary toform a homogenous solution. Then a solution of complex 4 in acetone wasadded to a solution of PUFA via the cannula and reaction was leftstirring at room temperature. Upon completion of the reaction, excess of2 N HCl (not less than 5 times volume of reaction mixture) was added andthe mixture was allowed to stir vigorously for 15 minutes. The productwas extracted with 100 ml of hexane and the solution was then washedwith saturated NaHCO₃ and NaCl solutions and dried over anhydrous NaSO₄.The solution was filtered and activated carbon was added. Stirring foranother 15 minutes, filtration and removal of volatiles in vacuoafforded desired product.

Synthesis of Deuterated Ethyl Linolenate (E-Lnn)

General procedure B was followed by mixing together 100 mg of E-Lnn(0.326 mmol), 1.18 ml of D₂O (65.40 mmol) and 1.42 mg of complex 4 (1%,3.26 μmol) in 10 ml of acetone and stirring for 1 hour to afford desireddeuterated product as clear colorless oil (101.06 mg, 99.6% yield).

Synthesis of Deuterated Ethyl Arachidonate (E-Ara)

General procedure B was followed by mixing together 100 mg of E-Ara(0.301 mmol), 1.63 ml of D₂O (90.22 mmol) and 1.31 mg of complex 4 (1%,3.01 μmol) in 12.5 ml of acetone and stirring for 24 hours to afforddesired deuterated product as clear colourless oil (101.36 mg, 99.5%yield).

Synthesis of Deuterated Ethyl Docosahexaenoate (E-DHA)

General procedure B was followed by mixing together 100 mg of E-DHA(0.280 mmol), 2.53 ml of D₂O (0.14 mol) and 2.44 mg of complex 4 (2%,5.61 μmol) in 15 ml of acetone and stirring for 18 hours to afforddesired deuterated product as clear colourless oil (101.96 mg, 99.8%yield).

Synthesis of deuterated trilinolenin (T-Lnn)

General procedure B was followed by mixing together 100 mg of T-Lnn(0.115 mmol), 1.24 ml of D₂O (68.70 mmol) and 0.50 mg of complex 4 (1%,1.15 mmol) in 20 ml of acetone and stirring for 7 hours to afforddesired deuterated product as clear colourless oil (101.34 mg, 99.7%yield).

CONCLUSION

While the disclosure has been illustrated and described in detail in thedrawings and foregoing description, such illustration and descriptionare to be considered illustrative or exemplary and not restrictive. Thedisclosure is not limited to the disclosed embodiments. Variations tothe disclosed embodiments can be understood and effected by thoseskilled in the art in practicing the claimed disclosure, from a study ofthe drawings, the disclosure and the appended claims.

All references cited herein are incorporated herein by reference intheir entirety. To the extent publications and patents or patentapplications incorporated by reference contradict the disclosurecontained in the specification, the specification is intended tosupersede and/or take precedence over any such contradictory material.

Unless otherwise defined, all terms (including technical and scientificterms) are to be given their ordinary and customary meaning to a personof ordinary skill in the art, and are not to be limited to a special orcustomized meaning unless expressly so defined herein. It should benoted that the use of particular terminology when describing certainfeatures or aspects of the disclosure should not be taken to imply thatthe terminology is being re-defined herein to be restricted to includeany specific characteristics of the features or aspects of thedisclosure with which that terminology is associated. Terms and phrasesused in this application, and variations thereof, especially in theappended claims, unless otherwise expressly stated, should be construedas open ended as opposed to limiting. As examples of the foregoing, theterm ‘including’ should be read to mean ‘including, without limitation,’‘including but not limited to,’ or the like; the term ‘comprising’ asused herein is synonymous with ‘including,’ ‘containing,’ or‘characterized by,’ and is inclusive or open-ended and does not excludeadditional, unrecited elements or method steps; the term ‘having’ shouldbe interpreted as ‘having at least;’ the term ‘includes’ should beinterpreted as ‘includes but is not limited to;’ the term ‘example’ isused to provide exemplary instances of the item in discussion, not anexhaustive or limiting list thereof; adjectives such as ‘known’,‘normal’, ‘standard’, and terms of similar meaning should not beconstrued as limiting the item described to a given time period or to anitem available as of a given time, but instead should be read toencompass known, normal, or standard technologies that may be availableor known now or at any time in the future; and use of terms like‘preferably,’‘preferred,’ ‘desired,’ or ‘desirable,’ and words ofsimilar meaning should not be understood as implying that certainfeatures are critical, essential, or even important to the structure orfunction of the invention, but instead as merely intended to highlightalternative or additional features that may or may not be utilized in aparticular embodiment of the invention. Likewise, a group of itemslinked with the conjunction ‘and’ should not be read as requiring thateach and every one of those items be present in the grouping, but rathershould be read as ‘and/or’ unless expressly stated otherwise. Similarly,a group of items linked with the conjunction ‘or’ should not be read asrequiring mutual exclusivity among that group, but rather should be readas ‘and/or’ unless expressly stated otherwise.

Where a range of values is provided, it is understood that the upper andlower limit, and each intervening value between the upper and lowerlimit of the range is encompassed within the embodiments.

With respect to the use of substantially any plural and/or singularterms herein, those having skill in the art can translate from theplural to the singular and/or from the singular to the plural as isappropriate to the context and/or application. The varioussingular/plural permutations may be expressly set forth herein for sakeof clarity. The indefinite article “a” or “an” does not exclude aplurality. A single processor or other unit may fulfill the functions ofseveral items recited in the claims. The mere fact that certain measuresare recited in mutually different dependent claims does not indicatethat a combination of these measures cannot be used to advantage. Anyreference signs in the claims should not be construed as limiting thescope.

It will be further understood by those within the art that if a specificnumber of an introduced claim recitation is intended, such an intentwill be explicitly recited in the claim, and in the absence of suchrecitation no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases “at least one” and “one or more” to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles “a” or “an” limits any particular claim containing suchintroduced claim recitation to embodiments containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (e.g., “a” and/or “an” should typically be interpreted to mean “atleast one” or “one or more”); the same holds true for the use ofdefinite articles used to introduce claim recitations. In addition, evenif a specific number of an introduced claim recitation is explicitlyrecited, those skilled in the art will recognize that such recitationshould typically be interpreted to mean at least the recited number(e.g., the bare recitation of “two recitations,” without othermodifiers, typically means at least two recitations, or two or morerecitations). Furthermore, in those instances where a conventionanalogous to “at least one of A, B, and C, etc.” is used, in generalsuch a construction is intended in the sense one having skill in the artwould understand the convention (e.g., “a system having at least one ofA, B, and C” would include but not be limited to systems that have Aalone, B alone, C alone, A and B together, A and C together, B and Ctogether, and/or A, B, and C together, etc.). In those instances where aconvention analogous to “at least one of A, B, or C, etc.” is used, ingeneral such a construction is intended in the sense one having skill inthe art would understand the convention (e.g., “a system having at leastone of A, B, or C” would include but not be limited to systems that haveA alone, B alone, C alone, A and B together, A and C together, B and Ctogether, and/or A, B, and C together, etc.). It will be furtherunderstood by those within the art that virtually any disjunctive wordand/or phrase presenting two or more alternative terms, whether in thedescription, claims, or drawings, should be understood to contemplatethe possibilities of including one of the terms, either of the terms, orboth terms. For example, the phrase “A or B” will be understood toinclude the possibilities of “A” or “B” or “A and B.”

All numbers expressing quantities of ingredients, reaction conditions,and so forth used in the specification are to be understood as beingmodified in all instances by the term ‘about.’ Accordingly, unlessindicated to the contrary, the numerical parameters set forth herein areapproximations that may vary depending upon the desired propertiessought to be obtained. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of anyclaims in any application claiming priority to the present application,each numerical parameter should be construed in light of the number ofsignificant digits and ordinary rounding approaches.

Furthermore, although the foregoing has been described in some detail byway of illustrations and examples for purposes of clarity andunderstanding, it is apparent to those skilled in the art that certainchanges and modifications may be practiced. Therefore, the descriptionand examples should not be construed as limiting the scope of theinvention to the specific embodiments and examples described herein, butrather to also cover all modification and alternatives coming with thetrue scope and spirit of the invention.

1. A compound of one of the formulae selected from:

wherein: R⁵ is an unsubstituted C₁-C₅ alkyl group; and each Y isindependently deuterium or tritium.
 2. The compound of claim 1, whereinthe compound has the formula


3. The compound of claim 1, wherein the compound has the formula


4. The compound of claim 1, wherein the compound has the formula


5. The compound of claim 1, wherein R⁵ is an unsubstituted ethyl.
 6. Thecompound of claim 1, wherein each Y is deuterium.
 7. The compound ofclaim 1, wherein R⁵ is an unsubstituted ethyl; and each Y is deuterium.8. The compound of claim 1, wherein the compound has an isotopic loadthat is in a range of from about 5% to about 50%.
 9. The compound ofclaim 1, wherein the compound has an isotopic load that is in a range offrom about 25% to about 50%.
 10. The compound of claim 1, wherein thecompound has an isotopic load that is in a range of from about 25% toabout 35%.
 11. A composition comprising one or more of a compound of theformulae selected from:

and one or both of eicosapentaenoic acid and docosahexaenoic acid,wherein: R⁵ is an unsubstituted C₁-C₅ alkyl group; and each Y isindependently deuterium or tritium.
 12. The composition of claim 11,wherein R⁵ is an unsubstituted ethyl.
 13. The composition of claim 11,wherein each Y is deuterium.
 14. The composition of claim 11, wherein R⁵is an unsubstituted ethyl; and each Y is deuterium.
 15. The compositionof claim 11, wherein the isotopic loads of the compounds areindependently in a range of from about 5% to about 50%.
 16. Thecomposition of claim 11, wherein the isotopic loads of the compounds areindependently in a range of from about 25% to about 50%.
 17. Thecomposition of claim 11, wherein the isotopic loads of the compounds areindependently in a range of from about 25% to about 35%.
 18. Thecomposition of claim 11, wherein the composition has an isotopic purityin a range of about 5% to about 99%.
 19. The composition of claim 11,wherein the composition has an isotopic purity in a range of about 5% toabout 50%.
 20. The composition of claim 11, wherein the composition hasan isotopic purity in a range of about 5% to about 25%.
 21. A compoundwhich is 7,7,10,10,13,13,16,16-D₈-eicosapentaenoic acid, or a salt orthereof:

is 6,6,9,9,12,12,15,15,18,18-D₁₀-docosahexaenoic acid, or a saltthereof:

or is 7,7,10,10,13,13-D₆-arachidonic acid,

or a salt thereof.
 22. The compound of claim 21, wherein the compoundis:

or a salt thereof.
 23. The compound of claim 21, wherein the compoundis:

or a salt thereof.
 24. The compound of claim 21, wherein the compoundis:

or a salt thereof.
 25. A composition comprising one, two, or three of(i) 7,7,10,10,13,13,16,16-D₈-eicosapentaenoic acid, or a salt orthereof:

(ii) 6,6,9,9,12,12,15,15,18,18-D₁₀-docosahexaenoic acid, or a saltthereof:

(iii) 7,7,10,10,13,13-D₆-arachadonic acid, or a salt thereof:

and (iv) one, two, or three of eicosapentaenoic acid, docosahexaenoicacid, and arachidonic acid, or a salt of any of the foregoing.
 26. Thecomposition of claim 25, wherein the composition comprises

or a salt thereof.
 27. The composition of claim 25, wherein thecomposition comprises

or a salt thereof.
 28. The composition of claim 25, wherein thecomposition comprises

or a salt thereof.
 29. The composition of claim 26, wherein thecomposition has an isotopic purity in a range of about 5% to about 99%.30. The composition of claim 26, wherein the composition has an isotopicpurity in a range of about 5% to about 50%.